Liquid cleaning compositions comprising protease variants

ABSTRACT

The present invention relates to compositions comprising protease variants suitable for use in cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/532,595filed Jun. 2, 2017, now allowed, which is a 35 U.S.C. 371 nationalapplication of PCT/EP2015/078588 filed Dec. 3, 2015, which claimspriority or the benefit under 35 U.S.C. 119 of European application nos.14196296.9 and 15194218.2, filed Dec. 4, 2014, and Nov. 12, 2015,respectively, the contents of which are fully incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compositions comprising proteasevariants suitable for use in, e.g., high pH cleaning or detergentcompositions, such as laundry detergent compositions and dish washcompositions, including automatic dish wash compositions.

Description of the Related Art

In the detergent industry, enzymes have for many decades beenimplemented in washing formulations. Enzymes used in such formulationscomprise amylases, cellulases, lipases, mannosidases, and proteases, aswell as other enzymes or mixtures thereof. Commercially the mostimportant enzymes are proteases. Many of the proteases traditionallyused in cleaning processes are alkaline proteases usually having a pHoptimum around 9 thus suitable for fairly alkaline detergents. However,some particular liquid detergents have a pH above 10 and thesedetergents have not previously been considered suitable for enzymes dueto the very harsh conditions and the resulting low storage stability ofenzymes in such detergents. In addition, enzymes tend to be less stablein liquid detergents in particular when the water content is high.

A number of useful protease variants have been described many of whichhave improved activity, stability, and solubility in differentdetergents

However, these variants are not suitable for very high pH detergent asthe alkaline environment of these detergents would make the enzymesdisintegrate and thus lower their activity after a very short time.

SUMMARY OF THE INVENTION

The present invention relates to protease variants stable under alkalineconditions and suitable for use in high pH liquid cleaning and detergentcompositions comprising such variants.

The present invention relates to a liquid cleaning composition having pH10 or above, comprising at least 0.01 wt % protease, wherein theprotease is a variant of a parent protease and wherein the proteasevariant has an amino acid sequence which has at least 60% sequenceidentity to the amino acid sequence of SEQ ID NO: 2 and wherein saidprotease variant is characterized by having at least 10% higher residualactivity than the parent protease when measured after 4 hours at 40° C.in liquid detergent with pH 10.

In another embodiment, the present invention relates to a liquidcleaning composition having pH 10 or above, comprising: (a) at least0.01 wt % protease, wherein the protease is a variant of a parentprotease and wherein the protease variant has an amino acid sequencewhich has at least 60% sequence identity to the amino acid sequence ofSEQ ID NO: 2 and wherein said protease variant is characterized byhaving at least 10% higher residual activity than the parent proteasewhen measured after 4 hours at 40° C. in liquid detergent with pH 10;and (b) optionally, a protease inhibitor, preferably said proteaseinhibitor is a peptide aldehyde protease inhibitor.

In another embodiment, the present invention relates to a liquidcleaning composition comprising: (a) from 20% to 95% wt, preferably from40% to 95% wt, further preferably from about 70% to about 90% wt water;(b) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10; and (c) optionally, a protease inhibitor,preferably said protease inhibitor is a peptide aldehyde proteaseinhibitor.

The invention further relates to a liquid cleaning compositioncomprising:

-   -   (a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergent        surfactant;    -   (b) from 40% to 95% wt, preferably from about 70% to about 90%        wt water;    -   (c) from 1% to 30% wt of an alkaline buffer system comprising an        alkali metal silicate or an alkali metal hydroxide or a mixture        thereof, to provide a pH of from about 11 to 13.5; and    -   (d) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a        protease variant.

In another embodiment, the present invention further relates to a liquidcleaning composition comprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant; and(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 11 to 13.5, preferably the pH is from about12 to 13.5;(e) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor.

In said embodiment, (c1) and (c2) can be used as alternatives to eachother or they can be used in combination with each other.

Definitions

The term “allelic variant” means any of two or more alternative forms ofa gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The term “cDNA” means a DNA molecule that can be prepared by reversetranscription from a mature, spliced, mRNA molecule obtained from aeukaryotic or prokaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps, including splicing, before appearing as mature spliced mRNA.

The term “coding sequence” means a polynucleotide, which directlyspecifies the amino acid sequence of a variant. The boundaries of thecoding sequence are generally determined by an open reading frame, whichbegins with a start codon such as ATG, GTG or TTG and ends with a stopcodon such as TAA, TAG, or TGA. The coding sequence may be a genomicDNA, cDNA, synthetic DNA, or a combination thereof.

The term “control sequences” means nucleic acid sequences necessary forexpression of a polynucleotide encoding a variant of the presentinvention. Each control sequence may be native (i.e., from the samegene) or foreign (i.e., from a different gene) to the polynucleotideencoding the variant or native or foreign to each other. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the polynucleotide encodinga variant.

The term “detergent component” is defined herein to mean the types ofchemicals which can be used in cleaning compositions such as detergentcompositions. Examples of detergent components are surfactants,hydrotropes, builders, co-builders, chelators or chelating agents,bleaching system or bleach components, polymers, fabric hueing agents,fabric conditioners, foam boosters, suds suppressors, dispersants, dyetransfer inhibitors, fluorescent whitening agents, perfume, opticalbrighteners, bactericides, fungicides, soil suspending agents, soilrelease polymers, anti-redeposition agents, enzyme inhibitors orstabilizers, enzyme activators, antioxidants, and solubilizers. Thedetergent composition may comprise of one or more of any type ofdetergent component.

The term “cleaning composition” includes “detergent composition” andincludes, unless otherwise indicated, all forms of cleaning compositionssuch as bar, homogenous tablet, tablet having two or more layers, pouchhaving one or more compartments, regular or compact powder, gel,granulate, liquid (e.g. regular, compact or concentrated liquid), paste,powder, spray or tablet compositions including heavy-duty liquids (HDL),fine-fabric liquid detergents, liquid and/or solid laundry detergentsand fine fabric detergents; hard surface cleaning formulations for,e.g., glass, wood, ceramic and metal counter tops and windows; carpetcleaners; oven cleaners; fabric fresheners; fabric softeners; textileand laundry pre-spotters, as well as dish wash detergents such as handdishwashing agents, light duty dishwashing agents, machine dishwashingagents; all-purpose or heavy-duty washing agents, liquid, gel orpaste-form all-purpose washing agents, liquid cleaning and disinfectingagents, including antibacterial hand-wash types, cleaning bars,mouthwashes, denture cleaners, car or carpet shampoos, bathroomcleaners; hair shampoos and hair-rinses; shower gels, foam baths; metalcleaners; as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types. Pouches can be configured as single ormulti-compartments. It can be of any form, shape and material which issuitable for hold the composition, e.g. without allowing the release ofthe composition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivatives thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blended compositions comprising hydrolytically degradableand water soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by MonoSol LLC,Indiana, USA) plus plasticisers like glycerol, ethylene glycerol,propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids: US2009/0011970 A1.

Detergent ingredients (or components) can be separated physically fromeach other by compartments in water dissolvable pouches or in differentlayers of tablets. Thereby negative storage interaction betweencomponents can be avoided. Different dissolution profiles of each of thecompartments can also give rise to delayed dissolution of selectedcomponents in the wash solution.

The cleaning composition (e.g. liquid cleaning composition) as describedherein is suitable for use in a cleaning process such as laundry or hardsurface cleaning including dish wash and industrial cleaning. Exemplarylaundry process may be selected from a group consisting of: residentiallaundry process, industrial laundry process and institutional laundryprocess.

In addition to containing a protease variant of the invention, thedetergent formulation may contain one or more additional enzymes (suchas, perhydrolases, amylases, catalases, cellulases (e.g.,endoglucanases), cutinases, haloperoxygenases, lipases, mannanases,pectinases, pectin lyases, peroxidases, proteases, DNases, xanthanases,and xyloglucanases, or any mixture thereof), and/or components such assurfactants, builders, chelators or chelating agents, bleach system orbleach components, polymers, fabric conditioners, foam boosters, sudssuppressors, dyes, perfume, tannish inhibitors, optical brighteners,bactericides, fungicides, soil suspending agents, anti-corrosion agents,enzyme inhibitors or stabilizers, enzyme activators, transferase(s),hydrolytic enzymes, oxidoreductases, bluing agents and fluorescent dyes,antioxidants, and solubilizers.

The term “dish wash” refers to all forms of washing dishes, e.g., byhand or automatic dish wash. Washing dishes includes, but is not limitedto, the cleaning of all forms of crockery such as plates, cups, glasses,bowls, all forms of cutlery such as spoons, knives, forks and servingutensils as well as ceramics, plastics such as melamine, metals, china,glass and acrylics.

The term “dish washing composition” refers to all forms of compositionsfor cleaning hard surfaces. The present invention is not restricted toany particular type of dish wash composition or any particulardetergent.

The term “expression” includes any step involved in the production of avariant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” means a linear or circular DNA moleculethat comprises a polynucleotide encoding a variant and is operablylinked to control sequences that provide for its expression.

The term “hard surface cleaning” is defined herein as cleaning of hardsurfaces wherein hard surfaces may include floors, tables, walls, roofsetc. as well as surfaces of hard objects such as cars (car wash) anddishes (dish wash). Dish washing includes but are not limited tocleaning of plates, cups, glasses, bowls, and cutlery such as spoons,knives, forks, serving utensils, ceramics, plastics such as melamine,metals, china, glass and acrylics.

The term “host cell” means any cell type that is susceptible totransformation, transfection, transduction, or the like with a nucleicacid construct or expression vector comprising a polynucleotide of thepresent invention. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication.

The term “improved property” means a characteristic associated with aprotease variant that is improved compared to the parent protease. Suchimproved properties include, but are not limited to, wash performance,protease activity, thermal activity profile, thermostability, pHactivity profile, pH stability, substrate/cofactor specificity, improvedsurface properties, substrate specificity, product specificity,increased stability, improved stability under storage conditions, andchemical stability.

The term “stability” includes storage stability and stability duringuse, e.g. during a wash process and reflects the stability of theprotease variant according to the invention as a function of time e.g.how much activity is retained when the protease variant is kept insolution in particular in a detergent solution. The stability isinfluenced by many factors e.g. pH, temperature, detergent compositione.g. amount of builder, surfactants etc. The protease stability may bemeasured using the assay described in example 3. The term “improvedstability” or “increased stability” is defined herein as a variantprotease displaying an increased stability in solutions, relative to thestability of the parent protease e.g. relative to SEQ ID NO: 2.

In a particular aspect of the invention, the improved stability is animproved stability in a liquid detergent having pH at 10 or above. Theterm “detergent stability” or “improved detergent stability is inparticular an improved stability of the protease activity when aprotease variant of the present invention is mixed into a liquiddetergent formulation having pH at 10 or above, and then stored at atemperature between 15 and 50° C., e.g., 20° C., 30° C. or 40° C.

The term “improved wash performance” is defined herein as a proteasevariant according to the invention displaying an improved washperformance relative to the wash performance of the parent protease,e.g., by increased stain removal. The term “wash performance” includeswash performance in laundry but also, e.g., in dish wash. The washperformance may be quantified as described under the definition of “washperformance” herein.

The term “isolated” means a substance in a form or environment whichdoes not occur in nature. Non-limiting examples of isolated substancesinclude (1) any non-naturally occurring substance, (2) any substanceincluding, but not limited to, any enzyme, variant, nucleic acid,protein, peptide or cofactor, that is at least partially removed fromone or more or all of the naturally occurring constituents with which itis associated in nature; (3) any substance modified by the hand of manrelative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

The term “laundering” relates to both household laundering andindustrial laundering and means a process of treating textiles and/orfabrics with a solution containing a detergent composition of thepresent invention. The laundering process can for example be carried outusing, e.g., a household or an industrial washing machine or can becarried out by hand.

The term “mature polypeptide” means a polypeptide in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, autocatalytic activation etc. In one aspect, the maturepolypeptide is amino acids 1 to 269 of SEQ ID NO: 2 and 1 to 275 of SEQID NO: 1. It is known in the art that a host cell may produce a mixtureof two of more different mature polypeptides (i.e., with a differentC-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide.

The term “mature polypeptide coding sequence” means a polynucleotidethat encodes a mature polypeptide having protease activity.

The term “nucleic acid construct” means a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or is modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature or which is synthetic, whichcomprises one or more control sequences.

The term “operably linked” means a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directsexpression of the coding sequence.

The term “parent” means a protease to which an alteration is made toproduce the enzyme variants of the present invention. It will beunderstood that in the present context the expression “having identicalamino acid sequence” relates to 100% sequence identity. In a particularembodiment the parent is a protease with at least 60% identity, such asat least 65%, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity toa polypeptide of SEQ ID NO: 1 or 2.

The term “protease” is defined herein as an enzyme that hydrolyzespeptide bonds. It includes any enzyme belonging to the EC 3.4 enzymegroup (including each of the thirteen subclasses thereof). The EC numberrefers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, SanDiego, Calif., including supplements 1-5 published in Eur. J. Biochem.1223: 1-5 (1994); Eur. J. Biochem. 232: 1-6 (1995); Eur. J. Biochem.237: 1-5 (1996); Eur. J. Biochem. 250: 1-6 (1997); and Eur. J. Biochem.264: 610-650 (1999); respectively. The most widely used proteases in thedetergent industry such as laundry and dish wash are the serineproteases or serine peptidases which is a subgroup of proteasescharacterised by having a serine in the active site, which forms acovalent adduct with the substrate. Further the subtilases (and theserine proteases) are characterized by having two active site amino acidresidues apart from the serine, namely a histidine residue and anaspartic acid residue. Subtilase refer to a sub-group of serine proteaseaccording to Siezen et al., 1991, Protein Engng. 4: 719-737 and Siezenet al., 1997, Protein Science 6: 501-523. The subtilases may be dividedinto 6 sub-divisions, i.e., the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family. The term “protease activity”means a proteolytic activity (EC 3.4). Proteases usably in detergentsare mainly endopeptidases (EC 3.4.21). There are several proteaseactivity types: The three main activity types are: trypsin-like wherethere is cleavage of amide substrates following Arg or Lys at P1,chymotrypsin-like where cleavage occurs following one of the hydrophobicamino acids at P1, and elastase-like with cleavage following an Ala atP1. For purposes of the present invention, protease activity isdetermined according to the Suc-AAPF-pNA activity assay, as described inthe Materials and Methods section below. In one aspect, the proteasevariants of the present invention have at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 100% of the enzyme activity of the maturepolypeptide of the parent enzyme. In one particular aspect the proteasevariants of the present invention have at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 100% of the enzyme activity of a polypeptide ofSEQ ID NO: 2.

The term “protease activity” means a proteolytic activity (EC 3.4).Proteases of the invention are endopeptidases (EC 3.4.21). There areseveral protease activity types: The three main activity types are:trypsin-like where there is cleavage of amide substrates following Argor Lys at P1, chymotrypsin-like where cleavage occurs following one ofthe hydrophobic amino acids at P1, and elastase-like with cleavagefollowing an Ala at P1. For purposes of the present invention, proteaseactivity is determined according to the procedure described in“Materials and Methods” below. The protease variants of the presentinvention preferably have at least 20%, e.g., at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, and at least 100% of the protease activity of a polypeptide of SEQID NO: 2.

The term “residual activity” means in this context the protease activityleft or remaining after storage and in particular after storage in aliquid detergent. When measuring the residual activity the activity ofthe protease is measured at t₁ just after adding the protease to thedetergent. The residual activity is then measured at t₂ after storage acertain amount of time, typical minutes or weeks. The protease variantof the invention preferably has residual activity of at least 10% atleast 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, or at least 100% of theprotease activity before storage (at t₁) in a high pH liquid detergentwith pH 10, at a temperature of 30° C. and when measured at t=4 hours orabove. A high pH liquid detergent is in this context a liquid detergentwith pH 10 or above, and a very high pH liquid detergent is in thiscontext a liquid detergent with pH 12 or above. A protease variantaccording to the invention is preferably more stable i.e. has higherresidual activity after storage in high pH liquid detergent (pH=10 ormore) compared to the parent protease e.g. a protease with SEQ ID NO 2and/or a protease variant according to the invention is more stable i.e.has higher residual activity after storage in liquid detergent i.e.liquid detergent comprising 40% water or above compared to the parentprotease e.g. a protease with SEQ ID NO 2. Preferably the residualactivity of the protease variant of the invention is at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45% such as at least 50% higher compared to theresidual activity of the parent protease e.g. compared to SEQ ID NO 2when measured after storage 4 hours at 30° C. in a liquid detergent withpH 10 and/or liquid detergent with at least 40% water. As described inExample 3.

In particular, the relative stability of a protease variant according tothe invention and its corresponding parent protease, are different athigh pH, above or at pH 10 from their relative stabilities at neutralpH, above or at pH 7.

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “sequence identity”.For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labelled “longestidentity” (obtained using the −nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

The different stringency conditions are defined as follows.

The term “substantially pure variant” means a preparation that containsat most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%,at most 2%, at most 1%, and at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated.Preferably, the variant is at least 92% pure, e.g., at least 94% pure,at least 95% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, at least 99.5% pure, and 100% pure by weight of thetotal polypeptide material present in the preparation. The variants ofthe present invention are preferably in a substantially pure form. Thiscan be accomplished, for example, by preparing the variant by well-knownrecombinant methods or by classical purification methods.

The term “substantially pure polynucleotide” means a polynucleotidepreparation free of other extraneous or unwanted nucleotides and in aform suitable for use within genetically engineered polypeptideproduction systems. Thus, a substantially pure polynucleotide containsat most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%,at most 2%, at most 1%, and at most 0.5% by weight of otherpolynucleotide material with which it is natively or recombinantlyassociated. A substantially pure polynucleotide may, however, includenaturally occurring 5′- and 3′-untranslated regions, such as promotersand terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, e.g., at least 92% pure, at least94% pure, at least 95% pure, at least 96% pure, at least 97% pure, atleast 98% pure, at least 99% pure, and at least 99.5% pure by weight.The polynucleotides of the present invention are preferably in asubstantially pure form.

The term “textile” means any textile material including yarns, yarnintermediates, fibers, non-woven materials, natural materials, syntheticmaterials, as well as fabrics made of these materials such as garments,cloths and other articles). When the term fabric or garment is used itis intended to include the broader term textiles as well.

The term “variant” means a polypeptide having protease activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at three or more (e.g., several) positions. A substitutionmeans replacement of the amino acid occupying a position with adifferent amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding one or more (e.g.,several) amino acids, e.g., 1, 2, 3, 4 or 5 amino acids adjacent to andimmediately following the amino acid occupying a position. The termprotease variant may be a variant of a subtilase parent, i.e., aprotease variant is a subtilase which comprises alterations i.e., asubstitution, insertion, and/or deletion, at three or more (e.g.,several) positions compared to the parent protease.

The term “wash performance” is used as an enzyme's ability to removestains present on the object to be cleaned during, e.g., wash, such aslaundry or hard surface cleaning. The improvement in the washperformance may be quantified by calculating the so-called intensityvalue (Int) defined in the AMSA assay.

The term “wild-type protease” means a protease expressed by a naturallyoccurring organism, such as a bacterium, archaea, yeast, fungus, plantor animal found in nature. An example of a wild-type subtilase issubtilisin Savinase, i.e., amino acids 1 to 269 of SEQ ID NO: 2.

Conventions for Designation of Variants

For purposes of the present invention, subtilisin BPN′ (the sequence ofamino acids 1-275 of SEQ ID NO: 1 (Siezen et al., 1991, Protein Eng. 4:719-737)) is used to determine the corresponding amino acid residue inanother protease. The amino acid sequence of another protease is alignedwith the mature polypeptide disclosed in SEQ ID NO: 1, and based on thealignment, the amino acid position number corresponding to any aminoacid residue in the polypeptide disclosed in SEQ ID NO: 1 is determinedusing the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol. Biol. 48: 443-453) as implemented in the Needle program of theEMBOSS package (EMBOSS: The European Molecular Biology Open SoftwareSuite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version5.0.0 or later. The parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in anotherprotease can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26:1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 1 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions:

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine at position 226 with alanine is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411 Phe” or “G205R+S411F”, representingsubstitutions at positions 205 and 411 of glycine (G) with arginine (R)and serine (S) with phenylalanine (F), respectively. An “X” preceding aposition means that any original amino acid at the position may besubstituted.

Deletions:

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions:

The insertion of an additional amino acid residue such as, e.g., alysine after G195 may be indicated by: Gly195GlyLys or G195GK.Alternatively insertion of an additional amino acid residue such aslysine after G195 may be indicated by: *195aK. When more than one aminoacid residue is inserted, such as, e.g., a Lys and Ala after G195 thismay be indicated as: Gly195GlyLysAla or G195GKA. In such cases, theinserted amino acid residue(s) may also be numbered by the addition oflower case letters to the position number of the amino acid residuepreceding the inserted amino acid residue(s), in this example: *195aK*195bA. In the above example, the sequences 194 to 196 would thus be:

-   -   194 195 196

Subtilisin 309 A-G-L

-   -   194 195 195a 195b 196

Variant A-G-K-A-L

In cases where a substitution and an insertion occur at the sameposition, this may be indicated as S99SD+S99A or in short S99AD. Thesame modification may also be indicated as S99A+*99aD.

In cases where an amino acid residue identical to the existing aminoacid residue is inserted, it is clear that degeneracy in thenomenclature arises. If for example a glycine is inserted after theglycine in the above example this would be indicated by G195GG or*195GaG. The same actual change could just as well be indicated asA194AG or *194aG for the change from:

-   -   194 195 196

Subtilisin 309 A-G-L

to:

-   -   194 195 195a 196

Variant A-G-G-L

-   -   194 194a 195 196

Such instances will be apparent to the skilled person and the indicationG195GG and corresponding indications for this type of insertions arethus meant to comprise such equivalent degenerate indications.

Multiple Alterations:

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of arginine and glycine at positions 170 and 195 withtyrosine and glutamic acid, respectively. Alternatively multiplealterations may be separated be space or a comma, e.g., A170Y G195E orA170Y, G195E respectively.

Different Alterations:

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine at position 170 with tyrosine orglutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”,“Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Alternatively different alterations or optional substitutions may beindicated in brackets, e.g., Arg170[Tyr, Gly] or Arg170{Tyr, Gly} or inshort R170 [Y,G] or R170 {Y, G}.

DETAILED DESCRIPTION OF THE INVENTION

Alkaline Liquid detergents having high pH are widely used in cleaning,such as laundry and dish wash cleaning. Liquid detergents with elevatedpH are especially commonly used by consumers in North America. The highpH cleaning compositions are also used in industrial cleaning processes.

Alkaline detergents are liquids having detergent properties. The pH ofsuch detergents usually ranges in pH from 9 to 12.5. The high pHdetergents typically comprise components such as surfactants, buildersand bleach components and additionally they may also contain asignificant amount of water and alkalis such as NaOH, TSP (Trisodiumphosphate), ammonia, Sodium carbonate, Potassium hydroxide (KOH) thesealkalis are usually added in amount corresponding to 0.1 to 30 percentweight (wt).

Examples of commercial high pH detergents include but are not limited toArm & Hammer (Church & Dwight), Surf Sparkling Ocean (Unilever), CleanBurst, SUN Triple Clean Tropical breeze (SUN products), Purex Free andClear (Henkel) and Xtra Tropical Passion (Church & Dwight).

Adding enzymes to detergents are highly advantageous as the specificactivities of these enzymes effectively removes specific stains fromsurfaces such as textile and cutlery. However, the difficulty ofmaintaining acceptable enzyme stability in the high pH liquid detergentshas for many years prohibited inclusion of enzymes into thesedetergents.

The present invention relates high pH liquid cleaning compositionscomprising alkaline stable protease variants suitable for use in suchcompositions.

One aspect of the invention relates to a liquid cleaning compositioncomprising:

-   -   (a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergent        surfactant;    -   (b) from 40% to 95% wt, preferably from about 70% to about 90%        wt water;    -   (c) from 1% to 30% wt of an alkaline buffer system comprising an        alkali metal silicate or an alkali metal hydroxide or a mixture        thereof, to provide a pH of from about 11 to 13.5; and    -   (d) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a        protease variant.

In another embodiment, the present invention relates to a liquidcleaning composition comprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant; and(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 7.5 to 13.5, preferably the pH is from about10 to 13.5;(d) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor.

The compositions of the invention preferably contain alkaline buffersystems to provide a pH of at least about 7.5, at least about 8, atleast about 9, preferably pH 10 or above. Preferably the pH is fromabout 9 to about 13. In order to achieve the high pH it is necessary tohave present an alkali metal hydroxide especially sodium or potassiumhydroxide, normally in an amount of 0.1 to about 30% by weight(percentage by weight, abbreviated wt %) of the composition, andpreferably 1.0 to 2.5%, or higher amounts of a suitable alkali metalsilicate such as metal silicate, according to the desired pH for theproduct.

The composition of the invention preferably contain from 20% to 95% wt,preferably from 40% to 95% wt, further preferably from about 70% toabout 90% wt water. Preferably from about 75 to 80% wt water, preferablyfrom about 80 to 85% wt water, preferably from about 70-75% wt water orpreferably from about 75-80% wt water. Preferably the composition of theinvention comprises 10% wt or more water, such as at least 20% wt, atleast 25% wt, at least 30% wt, at least 35% wt, at least 40% wt, atleast 45% wt, at least 50% wt, at least 55% wt, at least 60% wt, atleast 65% wt, at least 70% wt, at least 75% wt, at least 80% wt, atleast 85% wt, at least 90% wt, or at least 95% wt or more but less than100% wt percent weight (wt) water.

The protease variants may be added to a high pH cleaning compositionaccording to the invention in an amount corresponding to 0.01-200 mg ofenzyme protein per liter of wash liquor, preferably 0.05-50 mg of enzymeprotein per liter of wash liquor, in particular 0.1-10 mg of enzymeprotein per liter of wash liquor.

A composition for use in laundry liquid, for example, may include0.0001%-10%, such as 0.001-7%, such as 0.1%-5% of protease variantaccording to the invention by weight of the composition.

A composition for use in automatic dishwash (ADW), for example, mayinclude 0.0001%-50%, such as 0.001%-30%, such as 0.01%-20%, such as0.5-15% of enzyme protein by weight of the composition.

The present invention relates to a liquid cleaning composition having pH10 or above, comprising at least 0.01 wt % protease, wherein theprotease is a variant of a parent protease and wherein the proteasevariant has an amino acid sequence which has at least 60% sequenceidentity to the amino acid sequence of SEQ ID NO: 2, and wherein saidprotease variant is characterized by having at least 10% higher residualactivity than the parent protease when measured after 4 hours at 40° C.in liquid detergent with pH 10.

The protease variants added to a cleaning composition according to theinvention display half-lives that are improved to an extent that itenables the storage and use of proteases in high pH (pH>10.0) liquidcleaning compositions such as detergents. In addition the proteasevariants display significant wash performance in high pH liquids and ittherefore opens up the possibility of improving the wash performance ofhigh pH liquid detergents and/or reducing the concentration of detergentpresent in these detergent formulations to achieve the same washperformance. In addition the protease variants display significant washperformance in high pH liquids and it therefore opens up the possibilityof improving the wash performance of high pH liquid detergents and/orreducing the concentration of protease present in these detergentformulations to achieve the same wash performance.

One embodiment of the invention relates to a liquid cleaning compositionhaving pH 10 or above comprising a protease wherein the protease is avariant protease having at least 60% identity with the amino acidsequence of SEQ ID NO: 1 or 2, wherein said variant comprises asubstitution at one or more positions selected from the positionscorresponding to the positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182,185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260,261 and 262 of BPN′ (SEQ ID NO 1). In another embodiment, the inventionprovides a liquid cleaning composition having pH 10 or above andcomprising a protease wherein the protease is a variant protease havingat least 60% identity with the amino acid sequence of SEQ ID NO: 1 or 2,wherein said variant comprises substitutions at positions correspondingto the positions 205 and 209 of BPN′ (SEQ ID NO: 1), preferably saidvariant comprises substitutions at positions corresponding to thepositions 205 and 209 of BPN′ (SEQ ID NO: 1) and a substitution at oneor more positions selected from the positions corresponding to thepositions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206,216, 217, 218, 222, 255, 256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO:1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 7 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises a substitution at one or more positions selected fromthe positions corresponding to the positions 3, 9, 18, 43, 49, 61, 76,115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255,256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1). In anotherembodiment, the invention relates to a liquid cleaning compositionhaving pH from about 7 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises substitutions at positions corresponding to thepositions 205 and 209 of BPN′ (SEQ ID NO: 1), preferably said variantcomprises substitutions at positions corresponding to the positions 205and 209 of BPN′ (SEQ ID NO: 1) and a substitution at one or morepositions selected from the positions corresponding to the positions 3,9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218,222, 255, 256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 8 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises a substitution at one or more positions selected fromthe positions corresponding to the positions 3, 9, 18, 43, 49, 61, 76,115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255,256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1). In anotherembodiment, the invention relates to a liquid cleaning compositionhaving pH from about 8 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises substitutions at positions corresponding to thepositions 205 and 209 of BPN′ (SEQ ID NO: 1), preferably said variantcomprises substitutions at positions corresponding to the positions 205and 209 of BPN′ (SEQ ID NO: 1) and a substitution at one or morepositions selected from the positions corresponding to the positions 3,9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218,222, 255, 256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 9 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises a substitution at one or more positions selected fromthe positions corresponding to the positions 3, 9, 18, 43, 49, 61, 76,115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255,256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1). In anotherembodiment, the invention relates to a liquid cleaning compositionhaving pH from about 9 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises substitutions at positions corresponding to thepositions 205 and 209 of BPN′ (SEQ ID NO: 1), preferably said variantcomprises substitutions at positions corresponding to the positions 205and 209 of BPN′ (SEQ ID NO: 1) and a substitution at one or morepositions selected from the positions corresponding to the positions 3,9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218,222, 255, 256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 10 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises a substitution at one or more positions selected fromthe positions corresponding to the positions 3, 9, 18, 43, 49, 61, 76,115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255,256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1). In anotherembodiment, the invention relates to a liquid cleaning compositionhaving pH from about 10 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises substitutions at positions corresponding to thepositions 205 and 209 of BPN′ (SEQ ID NO: 1), preferably said variantcomprises substitutions at positions corresponding to the positions 205and 209 of BPN′ (SEQ ID NO: 1) and a substitution at one or morepositions selected from the positions corresponding to the positions 3,9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 206, 216, 217, 218,222, 255, 256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 11 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises a substitution at one or more positions selected fromthe positions corresponding to the positions 3, 9, 18, 43, 49, 61, 76,115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255,256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO 1). In another embodiment,the invention relates to a liquid cleaning composition having pH fromabout 11 to about 13 or above comprising a protease wherein the proteaseis a variant protease having at least 60% identity with the amino acidsequence of SEQ ID NO: 1 or 2, and wherein said variant comprisessubstitutions at positions corresponding to the positions 205 and 209 ofBPN′ (SEQ ID NO: 1), preferably said variant comprises substitutions atpositions corresponding to the positions 205 and 209 of BPN′ (SEQ IDNO: 1) and a substitution at one or more positions selected from thepositions corresponding to the positions 3, 9, 18, 43, 49, 61, 76, 115,120, 182, 185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260,261 and 262 of BPN′ (SEQ ID NO: 1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 7 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises one or more substitution corresponding to thesubstitutions X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W,X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P,X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D,X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262Dof BPN′ (SEQ ID NO: 1). In another embodiment, the invention relates toa liquid cleaning composition having pH from about 7 to about 13 orabove comprising a protease wherein the protease is a variant proteasehaving at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions correspondingto the substitutions X205I and X209W of BPN′ (SEQ ID NO: 1), preferablysaid variant comprises substitutions corresponding to the substitutionsX205I and X209W of BPN′ (SEQ ID NO: 1) and one or more substitutionscorresponding to the substitutions X3V, X9D, X9E, X18S, X43R, X43K,X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E,X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E,X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W,X262E and X262D of BPN′ (SEQ ID NO: 1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 8 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises one or more substitution corresponding to thesubstitutions X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W,X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P,X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D,X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262Dof BPN′ (SEQ ID NO: 1). In another embodiment, the invention relates toa liquid cleaning composition having pH from about 8 to about 13 orabove comprising a protease wherein the protease is a variant proteasehaving at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions correspondingto the substitutions X205I and X209W of BPN′ (SEQ ID NO: 1), preferablysaid variant comprises substitutions corresponding to the substitutionsX205I and X209W of BPN′ (SEQ ID NO: 1) and one or more substitutionscorresponding to the substitutions X3V, X9D, X9E, X18S, X43R, X43K,X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E,X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E,X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W,X262E and X262D of BPN′ (SEQ ID NO: 1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 9 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises one or more substitution corresponding to thesubstitutions X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W,X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P,X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D,X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262Dof BPN′ (SEQ ID NO: 1). In another embodiment, the invention relates toa liquid cleaning composition having pH from about 9 to about 13 orabove comprising a protease wherein the protease is a variant proteasehaving at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions correspondingto the substitutions X205I and X209W of BPN′ (SEQ ID NO: 1), preferablysaid variant comprises substitutions corresponding to the substitutionsX205I and X209W of BPN′ (SEQ ID NO: 1) and one or more substitutionscorresponding to the substitutions X3V, X9D, X9E, X18S, X43R, X43K,X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E,X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E,X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W,X262E and X262D of BPN′ (SEQ ID NO: 1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 10 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises one more substitution corresponding to thesubstitutions X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W,X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P,X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D,X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262Dof BPN′ (SEQ ID NO: 1). In another embodiment, the invention relates toa liquid cleaning composition having pH from about 10 to about 13 orabove comprising a protease wherein the protease is a variant proteasehaving at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions correspondingto the substitutions X205I and X209W of BPN′ (SEQ ID NO: 1), preferablysaid variant comprises substitutions corresponding to the substitutionsX205I and X209W of BPN′ (SEQ ID NO: 1) and one or more substitutionscorresponding to the substitutions X3V, X9D, X9E, X18S, X43R, X43K,X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E,X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E,X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W,X262E and X262D of BPN′ (SEQ ID NO: 1).

One embodiment of the invention relates to a liquid cleaning compositionhaving pH from about 11 to about 13 or above comprising a proteasewherein the protease is a variant protease having at least 60% identitywith the amino acid sequence of SEQ ID NO: 1 or 2, and wherein saidvariant comprises one more substitution corresponding to thesubstitutions X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W,X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P,X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D,X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262Dof BPN′ (SEQ ID NO: 1). In another embodiment, the invention relates toa liquid cleaning composition having pH from about 11 to about 13 orabove comprising a protease wherein the protease is a variant proteasehaving at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 2, and wherein said variant comprises substitutions correspondingto the substitutions X205I and X209W of BPN′ (SEQ ID NO: 1), preferablysaid variant comprises substitutions corresponding to the substitutionsX205I and X209W of BPN′ (SEQ ID NO: 1) and one or more substitutionscorresponding to the substitutions X3V, X9D, X9E, X18S, X43R, X43K,X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E,X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S, X255E,X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W,X262E and X262D of BPN′ (SEQ ID NO: 1).

In some preferred embodiments, the protease variant is selected from thegroup consisting of:

S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+

S256D+N261W+L262E;S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E.

In some preferred embodiments, the cleaning compositions provided hereinare formulated such that it has a pH of from about 7.5 to about 13.5,about 8.0 to about 13.5, about 8.5 to about 13.5, about 9.0 to about13.5, or in alternative embodiments, even from about 9.5 to about 13.5,such as from about 10 to about 13.5, from about 10 to about 13, fromabout 10 to about 12.5, from about 10 to about 12, from about 10 toabout 11.5, from about 10 to about 11, from about 10 to about 10.5. Insome preferred embodiments, the liquid cleaning composition isformulated such that pH is from about 10 to about 13.5. In otherpreferred embodiments, the liquid cleaning composition is formulatedsuch that pH is from about 12 to 13.5.

One aspect of the invention relates to a liquid cleaning compositioncomprising:

-   -   (a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergent        surfactant;    -   (b) from 40% to 95% wt, preferably from about 70% to about 90%        wt water;    -   (c) from 1% to 30% wt of an alkaline buffer system comprising an        alkali metal silicate or an alkali metal hydroxide or a mixture        thereof, to provide a pH of from about 11 to 13.5; and    -   (d) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a        protease variant, wherein said variant comprises a substitution        at one or more positions selected from the positions        corresponding to the positions 3, 9, 18, 43, 49, 61, 76, 115,        120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255,        256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1).

Another aspect of the invention relates to a liquid cleaning compositioncomprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;

(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant, wherein said variant comprises a substitution at one or morepositions selected from the positions corresponding to the positions 3,9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216,217, 218, 222, 255, 256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1);and

(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or

(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 8 to 13.5, preferably the pH is from about 10to 13.5;

(d) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor.

In a preferred embodiment, the protease variant comprises thesubstitutions X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W,X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P,X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D,X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D.Thus, one embodiment of the invention relates to a liquid cleaningcomposition comprising:

-   -   (a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergent        surfactant;    -   (b) from 40% to 95% wt, preferably from about 70% to about 90%        wt water;    -   (c) from 1% to 30% wt of an alkaline buffer system comprising an        alkali metal silicate or an alkali metal hydroxide or a mixture        thereof, to provide a pH of from about 11 to 13.5; and    -   (d) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a        protease variant, wherein said variant comprises one more        substitution corresponding to the substitutions X3V, X9D, X9E,        X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D,        X182D, X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L,        X209W, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E,        X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D        of BPN′ (SEQ ID NO 1), preferably said variant comprises        substitutions corresponding to the substitutions X205I and X209W        of BPN′ (SEQ ID NO 1), further preferably said variant comprises        substitutions corresponding to the substitutions X205I and X209W        of BPN′ (SEQ ID NO 1) and one or more substitutions        corresponding to the substitutions X3V, X9D, X9E, X18S, X43R,        X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D,        X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M,        X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A,        X260E, X260D, X261M, X261W, X262E and X262D of BPN′ (SEQ ID NO        1).

Another embodiment of the invention relates to a liquid cleaningcomposition comprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;

(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant, wherein said variant comprises one more substitutioncorresponding to the substitutions X3V, X9D, X9E, X18S, X43R, X43K,X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E,X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T,X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D,X261M, X261W, X262E and X262D of BPN′ (SEQ ID NO: 1), preferably saidvariant comprises substitutions corresponding to the substitutions X205Iand X209W of BPN′ (SEQ ID NO: 1), further preferably said variantcomprises substitutions corresponding to the substitutions X205I andX209W of BPN′ (SEQ ID NO: 1) and one or more substitutions correspondingto the substitutions X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D,X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D,X194P, X206L, X216V, X217M, X218T, X222S, X255E, X255D, X256D, X256E,X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E and X262D of BPN′(SEQ ID NO: 1); and

(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or

(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 8 to 13.5, preferably the pH is from about 10to 13.5;

(d) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor.

In some preferred embodiments, the protease variant is selected from thegroup consisting of:

S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E.

The protease variants of the present invention have improved stabilityin liquid cleaning composition at pH 10 and above when compared to theparent protease such as a protease with SEQ ID NO: 2. This makes thesevariants suitable for use in high pH liquid detergents for e.g. laundryor dish wash.

In one embodiment, the protease variant has improved stability, inparticular improved storage stability in a high pH liquid cleaningcomposition, compared to the parent protease. In a preferred embodiment,the protease variant has improved stability, in particular improvedstorage stability, and on par or improved wash performance compared tothe parent protease.

In an embodiment, the protease variant has improved storage stability ina high pH liquid detergent, and on par or improved wash performancecompared to the parent protease wherein storage stability may bemeasured using an ‘accelerated storage stability assay’ and washperformance may be measured using the Automatic Mechanical Stress Assay(AMSA).

In an embodiment, the protease variant has at least 60% but less than100% sequence identity to the parent protease. In an embodiment, theprotease variant has at least 70% but less than 100% sequence identityto the parent protease. In an embodiment, the protease variant has atleast 75% but less than 100% sequence identity to the parent protease.In an embodiment, the protease variant has at least 80% but less than100% sequence identity to the parent protease. In an embodiment, theprotease variant has at least 85% but less than 100% sequence identityto the parent protease. In an embodiment, the protease variant has atleast 90% but less than 100% sequence identity to the parent protease.In an embodiment, the protease variant has at least 93% but less than100% sequence identity to the parent protease.

In an embodiment, the protease variant has at least 95% but less than100% sequence identity to the parent protease. In an embodiment, theprotease variant has at least 96% but less than 100% sequence identityto the parent protease. In an embodiment, the protease variant has atleast 97% but less than 100% sequence identity to the parent protease.In an embodiment, the protease variant has at least 98% but less than100% sequence identity to the parent protease.

In an embodiment, the variant has an amino acid sequence which is atleast 60% identical to SEQ ID NO: 1, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has an amino acid sequence which isat least 60% identical to SEQ ID NO: 2, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 2.

In one aspect, the total number of alterations in the parent protease isbetween 3 and 30, preferably between 3 and 20, more preferably between 3and 15, even more preferably between 3 and 10, most preferably between 3and 8 alterations. In another aspect, total number of alterations in theparent protease is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 alterations.

The parent or the precursor protease may be any protease or even morepreferred any subtilase as defined below. The parent protease ispreferably a protease having at least 60% identity to SEQ ID NO: 2 butthe parent may be any serine protease. A serine protease is an enzymewhich catalyzes the hydrolysis of peptide bonds, and in which there isan essential serine residue at the active site (White, Handler andSmith, 1973 “Principles of Biochemistry,” Fifth Edition, McGraw-HillBook Company, NY, pp. 271-272). The bacterial serine proteases havemolecular weights in the 20,000 to 45,000 Dalton range. They areinhibited by diisopropylfluorophosphate. They hydrolyze simple terminalesters and are similar in activity to eukaryotic chymotrypsin, also aserine protease. Even more preferred parent is an alkaline protease,covering a sub-group, reflects the high pH optimum of some of the serineproteases, from pH 9.0 to 11.0 (for review, see Priest, 1977,Bacteriological Rev. 41: 711-753).

The parent protease may be a subtilase which is a sub-group of theserine proteases that has been proposed by Siezen et al., 1991, ProteinEng. 4:719-737 and Siezen et al., 1997, Protein Science 6:501-523. Theyare defined by homology analysis of more than 170 amino acid sequencesof serine proteases previously referred to as subtilisin-like proteases.A subtilisin was previously often defined as a serine protease producedby Gram-positive bacteria or fungi, and according to Siezen et al. nowis a subgroup of subtilases. A wide variety of subtilases have beenidentified, and the amino acid sequence of a number of subtilases hasbeen determined. For a more detailed description of such subtilases andtheir amino acid sequences reference is made to Siezen et al. (1997).The subtilisin is as mentioned a subgroup of subtilases which are serineproteases from the family S8, in particular from the subfamily S8A, asdefined by the MEROPS database(merops.sanger.ac.uk/cgi-bin/famsum?family=S8). Examples of subtilisinsare Subtilisin BPN′ (SEQ ID NO: 1) and subtilisin 309 (SEQ ID NO: 2)which have the MEROPS numbers S08.034 and S08.003, respectively. Aparent protease may also be a subtilase isolated from a natural source,wherein subsequent modifications (such as replacement(s) of the aminoacid side chain(s), substitution(s), deletion(s) and/or insertion(s))have been made while retaining the characteristic of a subtilase.Furthermore, a parent protease may be a subtilase which has beenprepared by the DNA shuffling technique, such as described by Ness etal., 1999, Nature Biotechnology, 17: 893-896.

Alternatively, the term “parent protease” may be termed “precursorprotease” and is used to describe the starting protease into whichmutations are made to obtain the variant of the invention. The parentprotease is preferably of the subtilisin subgroups.

One subgroup of the subtilases, I-S1 or “true” subtilisins, include the“classical” subtilisins, such as subtilisin 168 (BSS168), subtilisinBPN′, subtilisin Carlsberg (ALCALASE®, Novozymes A/S), and subtilisin DY(BSSDY). BPN′ is subtilisin BPN′ from B. amyloliquefaciens, SubtilisinBPN′ has the amino acid sequence of SEQ ID NO: 1. A further subgroup ofthe subtilases, I-S2 or high alkaline subtilisins, is recognized bySiezen et al. (supra). Sub-group I-S2 proteases are described as highlyalkaline subtilisins and include enzymes such as subtilisin PB92(BAALKP) (MAXACAL®, Genencor International Inc.), subtilisin 147(BLS147) (ESPERASE®, Novozymes A/S), alkaline elastase YaB (BSEYAB) andsubtilisin 309 (SAVINASE®, Novozymes A/S) having the amino acid sequenceSEQ ID NO: 2.

The homology between two amino acid sequences is in this contextdescribed by the parameter “identity” for purposes of the presentinvention, the degree of identity between two amino acid sequences isdetermined using the Needleman-Wunsch algorithm as described above. Theoutput from the routine is besides the amino acid alignment thecalculation of the “Percent Identity” between the two sequences.

Based on this description it is routine for a person skilled in the artto identify suitable homologous subtilases, which can be modifiedaccording to the invention.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 1, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 1. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 1.

The parent protease may be a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 2, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the polypeptide of SEQ ID NO: 2. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 2.

The parent protease may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent protease may be obtained from microorganisms of any genus.For purposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial protease. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces protease, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma protease.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis protease

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

A high pH stable protease variant according to the invention may beprepared by a method, comprising the steps of:

(a) introducing into a parent protease the substitutions X3V, X9D, X9E,X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D,X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V,X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A,X260E, X260D, X261M, X261W, X262E and X262D, wherein the positionscorresponds to the positions of SEQ ID NO: 1, and

(b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., US 2004/0171154; Storici et al., 2001,Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

The liquid cleaning compositions according the invention comprise inaddition to the proteases according to the invention additionalcomponents such as the non-limiting components set forth below. Thechoice of components may include, for fabric care, the consideration ofthe type of fabric to be cleaned, the type and/or degree of soiling, thetemperature at which cleaning is to take place, and the formulation ofthe detergent product.

In a particular embodiment, the high pH liquid cleaning compositioncomprises a protease variant of the invention and one or more detergentcomponents, such as surfactants, hydrotropes, builders, co-builders,chelators or chelating agents, bleaching system or bleach components,polymers, fabric hueing agents, fabric conditioners, foam boosters, sudssuppressors, dispersants, dye transfer inhibitors, fluorescent whiteningagents, perfume, optical brighteners, bactericides, fungicides, soilsuspending agents, soil release polymers, anti-redeposition agents,enzyme inhibitors or stabilizers, enzyme activators, antioxidants, andsolubilizers.

The protease variants of the cleaning composition of the invention maybe stabilized using conventional stabilizing agents and proteaseinhibitors, e.g., a polyol such as propylene glycol or glycerol, a sugaror sugar alcohol, different salts such as NaCl; KCl; lactic acid, formicacid, boric acid, or a boric acid derivative, e.g., an aromatic borateester, or a phenyl boronic acid derivative such as 4-formylphenylboronic acid (e.g. as in WO96/41859), or a peptide aldehyde such as di-,tri- or tetrapeptide aldehydes or aldehyde analogues (either of the formB1-B0-R wherein, R is H, CH3, CX3, CHX2, or CH2X (X=halogen), BO is asingle amino acid residue (preferably with an optionally substitutedaliphatic or aromatic side chain); and B1 consists of one or more aminoacid residues (preferably one, two or three), optionally comprising anN-terminal protection group (e.g. as in WO2009/118375, WO2013/004635),or as described in WO2005/105826, WO 2009/118375, WO 98/13459 or aprotease inhibitor of the protein type such as RASI, BASI, WASI(bifunctional alpha-amylase/subtilisin inhibitors of rice, barley andwheat) (e.g. as in WO2009/095425) or Cl2 or SSI.

Furthermore, the use of peptide aldehydes for stabilizing certainproteases in liquid detergents has been disclosed in PCT publicationsWO94/04651 and WO98/13460. More specifically, WO94/04651 discloses theuse of the peptide aldehydes Phe-Gly-Ala-PheH and Phe-Gly-Ala-LeuH forstabilizing subtilisin-type proteases. For stabilizing chymotrypsin-typeproteases, WO94/04651 discloses Leu-Leu-TyrH as a suitable peptidealdehyde. Furthermore, WO94/04651 proposes methyl carbamate or methylurea as an N-terminal protecting group of the peptide aldehydes.WO98/13460 discloses the use of peptide protease inhibitors, eitherpeptide aldehydes or trifluromethyl ketones, where the peptide chaincontains 2-5 amino acids and the aldehyde/trifluromethyl ketone isderived from the amino acids alanine, valine, isoleucine, leucine,phenylglycine, phenylalanine or homophenylalanine and where theN-terminal protection group is preferably a sulphonamide oramidophoshate. As an example CH3SO2Phe-Gly-Ala-LeuH can be used.

In another embodiment, a compound of the general formula I:

where R is selected from the group consisting of hydrogen, CH3, CX3,CHX2, CH2X, where X is a halogen atom, B1 is a single amino acid residueand B2 is one or more amino acid residues, B2 optionally comprising anN-terminal protection group, with the proviso that if B1 is leucine thenB2 cannot be leucine; can be used for stabilizing and/or inhibiting aprotease variant of the present invention.

In another embodiment, a protease inhibitor is a peptide compound of theformula B2-B1-B0-R wherein:

R is hydrogen, CH3, CX3, CHX2, or CH2X, wherein X is a halogen atom;B0 is a phenylalanine residue with an OH substituent at the p-positionand/or at the m-position;B1 is a single amino acid residue; andB2 consists of one or more amino acid residues, optionally comprising anN-terminal protection group.

Said protease inhibitor of the formula B2-B1-B0-R as described above canbe used as an optional protease inhibitor in the sense of the presentinvention (e.g. in combination with or as part of any embodiment of thepresent invention as disclosed herein)

Thus, in another embodiment the invention relates to a liquid cleaningcomposition having pH 10 or above, comprising:

(a) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10;

(b) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment, the invention relates to a liquid cleaningcomposition comprising:

(a) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water;

(b) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10;

(c) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment, the invention relates to a liquid cleaningcomposition comprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;

(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant; and

(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or

(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 11 to 13.5, preferably the pH is from about12 to 13.5;

(d) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In said embodiment, (c1) and (c2) can be used as alternatives or incombination with each other.

Thus, in another embodiment the invention relates to a liquid cleaningcomposition having pH 10 or above, comprising:

(a) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10, preferably said variant comprises a substitutionat one or more positions selected from the positions corresponding tothe positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194,205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262 ofBPN′ (SEQ ID NO 1);

(b) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment, the invention relates to a liquid cleaningcomposition comprising:

(a) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water;

(b) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10, preferably said variant comprises a substitutionat one or more positions selected from the positions corresponding tothe positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194,205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262 ofBPN′ (SEQ ID NO 1);

(c) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment, the invention relates to a liquid cleaningcomposition comprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;

(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant, preferably said variant comprises a substitution at one or morepositions selected from the positions corresponding to the positions 3,9, 18, 43, 49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216,217, 218, 222, 255, 256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO 1);and

(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or

(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 11 to 13.5, preferably the pH is from about12 to 13.5;

(d) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In said embodiment, (c1) and (c2) can be used as alternatives or incombination with each other.

Thus, in another embodiment the invention relates to a liquid cleaningcomposition having pH 10 or above, comprising:

(a) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10, preferably said variant comprises substitutions atpositions corresponding to the positions 205 and 209 of BPN′ (SEQ ID NO:1), further preferably said variant comprises substitutions at positionscorresponding to the positions 205 and 209 of BPN′ (SEQ ID NO: 1) and asubstitution at one or more positions selected from the positionscorresponding to the positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182,185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262of BPN′ (SEQ ID NO: 1);

(b) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment the invention relates to a liquid cleaningcomposition comprising:

(a) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water;

(b) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10, preferably said variant comprises substitutions atpositions corresponding to the positions 205 and 209 of BPN′ (SEQ ID NO:1), further preferably said variant comprises substitutions at positionscorresponding to the positions 205 and 209 of BPN′ (SEQ ID NO: 1) and asubstitution at one or more positions selected from the positionscorresponding to the positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182,185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262of BPN′ (SEQ ID NO 1);

(c) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment the invention relates to a liquid cleaningcomposition comprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;

(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant, preferably said variant comprises substitutions at positionscorresponding to the positions 205 and 209 of BPN′ (SEQ ID NO: 1),further preferably said variant comprises substitutions at positionscorresponding to the positions 205 and 209 of BPN′ (SEQ ID NO: 1) and asubstitution at one or more positions selected from the positionscorresponding to the positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182,185, 188, 194, 206, 216, 217, 218, 222, 255, 256, 259, 260, 261 and 262of BPN′ (SEQ ID NO: 1); and

(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or

(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 11 to 13.5, preferably the pH is from about12 to 13.5;

(d) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In said embodiment, (c1) and (c2) can be used as alternatives or incombination with each other.

Thus, in another embodiment the invention relates to a liquid cleaningcomposition having pH 10 or above, comprising:

(a) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10, preferably said variant comprises one moresubstitution corresponding to the substitutions X3V, X9D, X9E, X18S,X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E,X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M,X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E,X260D, X261M, X261W, X262E and X262D of BPN′ (SEQ ID NO 1);

(b) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment, the invention relates to a liquid cleaningcomposition comprising:

(d) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water;

(e) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10, preferably said variant comprises one moresubstitution corresponding to the substitutions X3V, X9D, X9E, X18S,X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E,X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M,X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E,X260D, X261M, X261W, X262E and X262D of BPN′ (SEQ ID NO 1);

(f) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment, the invention relates to a liquid cleaningcomposition comprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;

(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant, preferably said variant comprises one more substitutioncorresponding to the substitutions X3V, X9D, X9E, X18S, X43R, X43K,X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E, X185E,X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V, X217M, X218T,X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D,X261M, X261W, X262E and X262D of BPN′ (SEQ ID NO 1); and

(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or

(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 11 to 13.5, preferably the pH is from about12 to 13.5;

(d) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In said embodiment, (c1) and (c2) can be used as alternatives or incombination with each other.

Thus, in another embodiment the invention relates to a liquid cleaningcomposition having pH 10 or above, comprising:

(a) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10, preferably said variant comprises substitutionscorresponding to the substitutions X205I and X209W of BPN′ (SEQ ID NO1), further preferably said variant comprises substitutionscorresponding to the substitutions X205I and X209W of BPN′ (SEQ ID NO 1)and one or more substitutions corresponding to the substitutions X3V,X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V,X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V,X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A,X260E, X260D, X261M, X261W, X262E and X262D of BPN′ (SEQ ID NO 1);

(b) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment, the invention relates to a liquid cleaningcomposition comprising:

(a) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water;

(b) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10, preferably said variant comprises substitutionscorresponding to the substitutions X205I and X209W of BPN′ (SEQ ID NO1), further preferably said variant comprises substitutionscorresponding to the substitutions X205I and X209W of BPN′ (SEQ ID NO 1)and one or more substitutions corresponding to the substitutions X3V,X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V,X120D, X182D, X182E, X185E, X185D, X188E, X188D, X194P, X206L, X216V,X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A,X260E, X260D, X261M, X261W, X262E and X262D of BPN′ (SEQ ID NO 1);

(c) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In another embodiment, the invention relates to a liquid cleaningcomposition comprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;

(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant, preferably said variant comprises substitutions correspondingto the substitutions X205I and X209W of BPN′ (SEQ ID NO: 1), furtherpreferably said variant comprises substitutions corresponding to thesubstitutions X205I and X209W of BPN′ (SEQ ID NO 1) and one or moresubstitutions corresponding to the substitutions X3V, X9D, X9E, X18S,X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D, X182E,X185E, X185D, X188E, X188D, X194P, X206L, X216V, X217M, X218T, X222S,X255E, X255D, X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M,X261W, X262E and X262D of BPN′ (SEQ ID NO 1); and

(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or

(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 11 to 13.5, preferably the pH is from about12 to 13.5;

(d) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor, further preferably saidprotease inhibitor is a compound of the general formula I, where R isselected from the group consisting of hydrogen, CH3, CX3, CHX2, CH2X,where X is a halogen atom, B1 is a single amino acid residue and B2 isone or more amino acid residues, B2 optionally comprising an N-terminalprotection group, with the proviso that if B1 is leucine then B2 cannotbe leucine.

In said embodiment, (c1) and (c2) can be used as alternatives or incombination with each other.

In some preferred embodiments, the protease variant is selected from thegroup consisting of:

S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E.

The composition may be formulated as described in, e.g., WO 92/19709, WO92/19708 and U.S. Pat. No. 6,472,364. In some embodiments, the enzymesemployed herein are stabilized by the presence of water-soluble sourcesof zinc (II), calcium (II) and/or magnesium (II) ions in the finishedcompositions that provide such ions to the enzymes, as well as othermetal ions (e.g., barium (II), scandium (II), iron (II), manganese (II),aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II), andoxovanadium (IV).

A protease variant of the present invention may also be incorporated inthe detergent formulations disclosed in WO 97/07202, which is herebyincorporated by reference.

A liquid cleaning composition of the invention is characterised byhaving high pH (at or above pH 10). The liquid high pH cleaningcomposition may further comprise high amount of water. In one preferredaspect of the invention, the liquid cleaning composition comprises:

-   -   (a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergent        surfactant;    -   (b) from 40% to 95% wt, preferably from about 70% to about 90%        wt water;    -   (c) from 1% to 30% wt of an alkaline buffer system comprising an        alkali metal silicate or an alkali metal hydroxide or a mixture        thereof, to provide a pH of from about 11 to 13.5; and    -   (d) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a        protease variant.

The surfactants may be anionic and/or cationic and/or non-ionic and/orsemi-polar and/or zwitterionic, or a mixture thereof. In a particularembodiment, the cleaning composition includes a mixture of one or morenonionic surfactants and one or more anionic surfactants. Thesurfactant(s) is typically present at a level of from about 0.1% to 60%by weight, such as about 1% to about 40%, or about 3% to about 20%, orabout 3% to about 10%. When included in a cleaning composition accordingto the invention the surfactants will usually contain from about 1% toabout 40% by weight, such as from about 1% to about 20%, preferably fromabout 0% to about 5% by weight. Non-limiting examples of anionicsurfactants include sulfates and sulfonates, in particular, linearalkylbenzenesulfonates (LAS), isomers of LAS, branchedalkylbenzenesulfonates (BABS), phenylalkanesulfonates,alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates,alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcoholsulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates(AES or AEOS or FES, also known as alcohol ethoxysulfates or fattyalcohol ether sulfates), secondary alkanesulfonates (SAS), paraffinsulfonates (PS), ester sulfonates, sulfonated fatty acid glycerolesters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES)including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives ofamino acids, diesters and monoesters of sulfo-succinic acid or soap, andcombinations thereof.

When included the cleaning composition according to the invention itwill usually contain from about 0% to about 10% by weight of a cationicsurfactant. Non-limiting examples of cationic surfactants includealklydimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide(CTAB), dimethyldistearylammonium chloride (DSDMAC), andalkylbenzyldimethylammonium, alkyl quaternary ammonium compounds,alkoxylated quaternary ammonium (AQA) compounds, and combinationsthereof.

Non-limiting examples of non-ionic surfactants include alcoholethoxylates (AE or AEO), alcohol propoxylates, propoxylated fattyalcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylatedand/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates(APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG),alkoxylated amines, fatty acid monoethanolamides (FAM), fatty aciddiethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM),propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkylfatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

Non-limiting examples of semipolar surfactants include amine oxides (AO)such as alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxideand N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

Non-limiting examples of zwitterionic surfactants include betaine,alkyldimethylbetaine, sulfobetaine, and combinations thereof.

Particularly preferred surfactants include LAS, MES, alcohol ethersulfate, ethoxylated lauryl alcohol or sodium dodecylbenzenesulfonate.

A liquid high pH cleaning composition according to the invention,typically contain at least 20% by weight and up to 95% by weight water,such as up to about 70% wt water, up to about 65% wt water, up to about55% wt water, up to about 45% wt water, up to about 35% wt water. Othertypes of liquids, including without limitation, alkanols, amines, diols,ethers and polyols may be included in the liquid cleaning composition.The cleaning composition may contain from 0-30% organic solvent.

The cleaning composition may also contain a hydrotrope which is acompound that solubilizes hydrophobic compounds in aqueous solutions (oroppositely, polar substances in a non-polar environment). Typically,hydrotropes have both hydrophilic and hydrophobic characters (so-calledamphiphilic properties as known from surfactants); however the molecularstructure of hydrotropes generally do not favor spontaneousself-aggregation, see, e.g., review by Hodgdon and Kaler, 2007, CurrentOpinion in Colloid & Interface Science 12: 121-128. Hydrotropes do notdisplay a critical concentration above which self-aggregation occurs asfound for surfactants and lipids forming miceller, lamellar or otherwell defined meso-phases. Instead, many hydrotropes show acontinuous-type aggregation process where the sizes of aggregates growas concentration increases. However, many hydrotropes alter the phasebehavior, stability, and colloidal properties of systems containingsubstances of polar and non-polar character, including mixtures ofwater, oil, surfactants, and polymers. Hydrotropes are classically usedacross industries from pharma, personal care, food, to technicalapplications. Use of hydrotropes in cleaning compositions allows forexample more concentrated formulations of surfactants (as in the processof compacting liquid detergents by removing water) without inducingundesired phenomena such as phase separation or high viscosity. Thecleaning composition according to the invention may contain 0-5% byweight, such as about 0.5 to about 5%, or about 3% to about 5% wt, of ahydrotrope. Any hydrotrope known in the art for use in detergents may beutilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate(SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amineoxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodiumhydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, andcombinations thereof. The cleaning composition according to theinvention may contain about 0-65% by weight, such as about 5% to about45% of a detergent builder or co-builder, or a mixture thereof. In adish wash detergent, the level of builder is typically 40-65%,particularly 50-65%. Builders and chelators soften, e.g., the wash waterby removing the metal ions from the liquid. The builder and/orco-builder may particularly be a chelating agent that formswater-soluble complexes with Ca and Mg. Any builder and/or co-builderknown in the art for use in laundry detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, alsoknown as iminodiethanol), triethanolamine (TEA, also known as2,2′,2″-nitrilotriethanol), and carboxymethyl inulin (CMI), andcombinations thereof.

The cleaning composition according to the invention may also contain0-20% by weight, such as about 5% to about 10% wt, of a detergentco-builder, or a mixture thereof. The detergent composition may includea co-builder alone, or in combination with a builder, for example azeolite builder. Non-limiting examples of co-builders includehomopolymers of polyacrylates or copolymers thereof, such aspoly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA).Further non-limiting examples include citrate, chelators such asaminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- oralkenylsuccinic acid. Additional specific examples include2,2′,2″-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid(IDS), ethylenediamine-N,N′-disuccinic acid (EDDS),methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid(GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP),ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA),diethylenetriaminepentakis (methylenephosphonic acid) (DTPMPA or DTMPA),N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL),N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid(MIDA), α-alanine-N, N-diacetic acid (α-ALDA), serine-N, N-diacetic acid(SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, N-diaceticacid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilicacid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) andsulfomethyl-N, N-diacetic acid (SMDA),N-(2-hydroxyethyl)-ethylidenediamine-N, N′, N′-triacetate (HEDTA),diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonicacid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), andcombinations and salts thereof. Further exemplary builders and/orco-builders are described in, e.g., WO 2009/102854 and U.S. Pat. No.5,977,053.

The cleaning composition according to the invention may contain 0-50% byweight, such as about 0.1% to about 25%, of a bleaching system. Bleachsystems remove discolor often by oxidation, and many bleaches also havestrong bactericidal properties, and are used for disinfecting andsterilizing. Any bleaching system known in the art for use in laundrydetergents may be utilized. Suitable bleaching system components includebleaching catalysts, hypochlorite, photobleaches, bleach activators,sources of hydrogen peroxide such as sodium percarbonate and sodiumperborates, preformed peracids and mixtures thereof.

Some non-limiting examples of bleach catalysts that may be used in thecompositions of the present invention include manganese oxalate,manganese acetate, manganese-collagen, cobalt-amine catalysts andmanganese triazacyclononane (MnTACN) catalysts; particularly preferredare complexes of manganese with 1,4,7-trimethyl-1,4,7-triazacyclononane(Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), inparticular Me3-TACN, such as the dinuclear manganese complex[(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and[2,2′,2″-nitrilotris(ethane-1,2-diylazanylylidene-KN-methanylylidene)triphenolato-K3O]manganese(III).The bleach catalysts may also be other metal compounds, such as iron orcobalt complexes.

Suitable preformed peracids include, but are not limited to,peroxycarboxylic acids and salts, percarbonic acids and salts, perimidicacids and salts, peroxymonosulfuric acids and salts, for example, Oxone(R), and mixtures thereof. Non-limiting examples of bleaching systemsinclude peroxide-based bleaching systems, which may comprise, forexample, an inorganic salt, including alkali metal salts such as sodiumsalts of perborate (usually mono- or tetra-hydrate), percarbonate,persulfate, perphosphate, persilicate salts, in combination with aperacid-forming bleach activator. The term bleach activator is meantherein as a compound which reacts with peroxygen bleach like hydrogenperoxide to form a peracid. The peracid thus formed constitutes theactivated bleach. Suitable bleach activators to be used herein includethose belonging to the class of esters amides, imides or anhydrides.Suitable examples are tetracetylethylene diamine (TAED), sodium4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxydodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS),4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed in WO98/17767. A particular family of bleach activators was disclosed in EP624154 and particularly acetyl triethyl citrate (ATC). ATC or a shortchain triglyceride like triacetin has the advantage that it isenvironmental friendly as it eventually degrades into citric acid andalcohol. Furthermore, acetyl triethyl citrate and triacetin has a goodhydrolytical stability in the product upon storage and it is anefficient bleach activator. Finally ATC provides a good buildingcapacity to the laundry additive. Alternatively, the bleaching systemmay comprise peroxyacids of, for example, the amide, imide, or sulfonetype. The bleaching system may also comprise peracids such as6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching system may alsoinclude a bleach catalyst.

In some embodiments, the bleach component may be an organic catalystselected from the group consisting of organic catalysts having thefollowing formula:

(iii) and mixtures thereof; wherein each R¹ is independently a branchedalkyl group containing from 9 to 24 carbons or linear alkyl groupcontaining from 11 to 24 carbons, preferably each R¹ is independently abranched alkyl group containing from 9 to 18 carbons or linear alkylgroup containing from 11 to 18 carbons, more preferably each R¹ isindependently selected from the group consisting of 2-propylheptyl,2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl,n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl andiso-pentadecyl. Other exemplary bleaching systems are described, e.g.,in WO 2007/087258, WO 2007/087244, WO 2007/087259 and WO 2007/087242.Suitable photobleaches may for example be sulfonated zincphthalocyanine.

One particular bleaching agent is hypochlorite such as sodiumhypochlorite (NaClO). Hypochlorite is stabilized by high pH and inaddition hypochlorite is antibacterial. In general, a cleaningcomposition according to the invention contains 3-8% sodium hypochloriteand 0.01-0.05% sodium hydroxide; the sodium hydroxide is used to slowthe decomposition of sodium hypochlorite into sodium chloride and sodiumchlorate. The hypochlorite may not be part of a detergent formula but aseparate product added during the wash process.

In one aspect of the invention, a high pH cleaning composition of theinvention does not comprise any bleach agents i.e. the cleaningcomposition may be bleach free.

The cleaning composition according to the invention may contain 0-10% byweight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymerknown in the art for use in detergents may be utilized. The polymer mayfunction as a co-builder as mentioned above, or may provideantiredeposition, fiber protection, soil release, dye transferinhibition, grease cleaning and/or anti-foaming properties. Somepolymers may have more than one of the above-mentioned properties and/ormore than one of the below-mentioned motifs. Exemplary polymers include(carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA),poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethyleneoxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin(CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid,and lauryl methacrylate/acrylic acid copolymers, hydrophobicallymodified CMC (HM-CMC) and silicones, copolymers of terephthalic acid andoligomeric glycols, copolymers of poly(ethylene terephthalate) andpoly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole)(PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) andpolyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymersinclude sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated. The cleaningcompositions of the present invention may also include fabric hueingagents such as dyes or pigments, which when formulated in detergentcompositions can deposit onto a fabric when the fabric is contacted witha wash liquor comprising the detergent compositions and thus alteringthe tint of the fabric through absorption/reflection of visible light.Fluorescent whitening agents emit at least some visible light. Incontrast, fabric hueing agents alter the tint of a surface as theyabsorb at least a portion of the visible light spectrum. Suitable fabrichueing agents include dyes and dye-clay conjugates, and may also includepigments. Suitable dyes include small molecule dyes and polymeric dyes.Suitable small molecule dyes include small molecule dyes selected fromthe group consisting of dyes falling into the Colour Index (C.I.)classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue,Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof, for example as described in WO 2005/003274, WO2005/003275, WO 2005/003276 and EP 1876226 (hereby incorporated byreference). The detergent composition preferably comprises from about0.00003 wt. % to about 0.2 wt. %, from about 0.00008 wt. % to about 0.05wt. %, or even from about 0.0001 wt. % to about 0.04 wt. % fabric hueingagent. The composition may comprise from 0.0001 wt % to 0.2 wt. % fabrichueing agent, this may be especially preferred when the composition isin the form of a unit dose pouch. Suitable hueing agents are alsodisclosed in, e.g., WO 2007/087257 and WO 2007/087243.

The cleaning composition according to the invention may comprise one ormore (additional) enzymes such as an amylase, perhydrolase, arabinase,carbohydrase, cellulase (e.g., endoglucanase), cutinase, galactanase,haloperoxygenase, lipase, mannanase, oxidase, e.g., laccase and/orperoxidase, pectinase, pectin lyases, protease, DNases, xylanase,xanthanase, and xyloglucanase.

In general, the properties of the selected enzyme(s) should becompatible with detergent (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Commercially available cellulases includeCelluzyme™, and Carezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™(Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

The composition may comprise one or more additional proteases includingthose of bacterial, fungal, plant, viral or animal origin, e.g.,vegetable or microbial origin. Microbial origin is preferred. Chemicallymodified or protein engineered mutants are included. It may be analkaline protease, such as a serine protease or a metalloprotease. Aserine protease may for example be of the S1 family, such as trypsin, orthe S8 family such as subtilisin. A metalloproteases protease may forexample be a thermolysin from, e.g., family M4 or other metalloproteasesuch as those from M5, M7 or M8 families.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO 2007/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®,Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®,FN3®, FN4®, Excellase®, Eraser®, Opticlean® and Optimase®(Danisco/DuPont), Axapem™ (Gist-Brocades N.V.), BLAP (sequence shown inFIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) andKAP (Bacillus alkalophilus subtilisin) from Kao.

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g., from T.lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP 305216, cutinase from Humicola, e.g., H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamedto Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95/06720 & WO96/27002), P. wisconsinensis (WO 96/12012), GDSL-type Streptomyceslipases (WO 2010/065455), cutinase from Magnaporthe grisea (WO2010/107560), cutinase from Pseudomonas mendocina (U.S. Pat. No.5,389,536), lipase from Thermobifida fusca (WO 2011/084412), Geobacillusstearothermophilus lipase (WO 2011/084417), lipase from Bacillussubtilis (WO 2011/084599), and lipase from Streptomyces griseus (WO2011/150157) and S. pristinaespiralis (WO 2012/137147). Preferredcommercial lipase products include Lipolase™ Lipex™; Lipolex™ andLipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g., acyltransferases with homologyto Candida antarctica lipase A (WO 2010/111143), acyltransferase fromMycobacterium smegmatis (WO 2005/056782), perhydrolases from the CE 7family (WO 2009/067279), and variants of the M. smegmatis perhydrolasein particular the S54V variant used in the commercial product GentlePower Bleach from Huntsman Textile Effects Pte Ltd (WO 2010/100028).

Suitable amylases may be an alpha-amylase or a glucoamylase and may beof bacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Commercially available amylases are Duramyl™,Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, LiquozymeX and BAN™ (from Novozymes A/S), and Rapidase™ Purastar™/Effectenz™,Powerase, Preferenz S1000, Preferenz S100 and Preferenz 5110 (fromGenencor International Inc./DuPont).

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in the cleaning composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Any detergent components known in the art for use in laundry cleaningcompositions may also be utilized. Other optional detergent componentsinclude anti-corrosion agents, anti-shrink agents, anti-soilredeposition agents, anti-wrinkling agents, bactericides, binders,corrosion inhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

The cleaning compositions of the present invention can also containdispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms. Suitable dispersants are for exampledescribed in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc.

The cleaning compositions of the present invention may also include oneor more dye transfer inhibiting agents. Suitable polymeric dye transferinhibiting agents include, but are not limited to, polyvinylpyrrolidonepolymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidoneand N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a subject composition, the dyetransfer inhibiting agents may be present at levels from about 0.0001%to about 10%, from about 0.01% to about 5% or even from about 0.1% toabout 3% by weight of the composition.

The cleaning compositions of the present invention will preferably alsocontain additional components that may tint articles being cleaned, suchas fluorescent whitening agent or optical brighteners. Where present thebrightener is preferably at a level of about 0.01% to about 05%. Anyfluorescent whitening agent suitable for use in a laundry detergentcomposition may be used in the composition of the present invention. Themost commonly used fluorescent whitening agents are those belonging tothe classes of diaminostilbene-sulphonic acid derivatives,diarylpyrazoline derivatives and bisphenyl-distyryl derivatives.Examples of the diaminostilbene-sulphonic acid derivative type offluorescent whitening agents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate and2-(stilbyl-4″-naptho-1,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferredfluorescent whitening agents are Tinopal DMS and Tinopal CBS availablefrom Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium saltof 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbenedisulphonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use in the invention include the 1-3-diaryl pyrazolines andthe 7-alkylaminocoumarins. Suitable fluorescent brightener levelsinclude lower levels of from about 0.01, from 0.05, from about 0.1 oreven from about 0.2 wt. % to upper levels of 0.5 or even 0.75 wt. %.

The cleaning compositions of the present invention may also include oneor more soil release polymers which aid the removal of soils fromfabrics such as cotton and polyester based fabrics, in particular theremoval of hydrophobic soils from polyester based fabrics. The soilrelease polymers may for example be nonionic or anionic terephthaltebased polymers, polyvinyl caprolactam and related copolymers, vinylgraft copolymers, polyester polyamides see for example Chapter 7 inPowdered Detergents, Surfactant science series volume 71, Marcel Dekker,Inc. Another type of soil release polymers are amphiphilic alkoxylatedgrease cleaning polymers comprising a core structure and a plurality ofalkoxylate groups attached to that core structure. The core structuremay comprise a polyalkylenimine structure or a polyalkanolaminestructure as described in detail in WO 2009/087523 (hereby incorporatedby reference). Furthermore random graft co-polymers are suitable soilrelease polymers Suitable graft co-polymers are described in more detailin WO 2007/138054, WO 2006/108856 and WO 2006/113314 (herebyincorporated by reference). Other soil release polymers are substitutedpolysaccharide structures especially substituted cellulosic structuressuch as modified cellulose deriviatives such as those described in EP1867808 or WO 03/040279 (both are hereby incorporated by reference).Suitable cellulosic polymers include cellulose, cellulose ethers,cellulose esters, cellulose amides and mixtures thereof. Suitablecellulosic polymers include anionically modified cellulose, nonionicallymodified cellulose, cationically modified cellulose, zwitterionicallymodified cellulose, and mixtures thereof. Suitable cellulosic polymersinclude methyl cellulose, carboxy methyl cellulose, ethyl cellulose,hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, estercarboxy methyl cellulose, and mixtures thereof.

The cleaning compositions of the present invention may also include oneor more anti-redeposition agents such as carboxymethylcellulose (CMC),polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethyleneand/or polyethyleneglycol (PEG), homopolymers of acrylic acid,copolymers of acrylic acid and maleic acid, and ethoxylatedpolyethyleneimines. The cellulose based polymers described under soilrelease polymers above may also function as anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,and structurants for liquid detergents and/or structure elasticizingagents.

The cleaning composition of the invention may be formulated into poucheswhich can be configured as single or multicompartments. It can be of anyform, shape and material which is suitable for hold the composition,e.g., without allowing the release of the composition from the pouchprior to water contact. The pouch is made from water soluble film whichencloses an inner volume. The inner volume can be divided intocompartments of the pouch. Preferred films are polymeric materialspreferably polymers which are formed into a film or sheet. Preferredpolymers, copolymers or derivates thereof are selected polyacrylates,and water soluble acrylate copolymers, methyl cellulose, carboxy methylcellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, mostpreferably polyvinyl alcohol copolymers and, hydroxyprpyl methylcellulose (HPMC). Preferably the level of polymer in the film forexample PVA is at least about 60%. Preferred average molecular weightwill typically be about 20,000 to about 150,000. Films can also be ofblend compositions comprising hydrolytically degradable and watersoluble polymer blends such as polyactide and polyvinyl alcohol (knownunder the Trade reference M8630 as sold by Chris Craft In. Prod. ofGary, Ind., US) plus plasticisers like glycerol, ethylene glycerol,Propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry detergent composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids. See, e.g., US2009/0011970.

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

The present invention is also directed to methods for using the cleaningcompositions according to the invention in laundering of textile andfabrics, such as house hold laundry washing and industrial laundrywashing. Such methods includes methods for using the compositionsaccording to the invention or compositions thereof in cleaning hardsurfaces such as floors, tables, walls, roofs etc. as well as surfacesof hard objects such as cars (car wash) and dishes (dish wash).

The protease variants of the present invention may be added to and thusbecome a component of the high pH cleaning composition according to theinvention. Thus one aspect of the invention relates to the use of aprotease variant in a high pH cleaning process such as laundering and/orhard surface cleaning at pH at or above pH 10.

A cleaning composition of the present invention may be formulated, forexample, as a hand or machine laundry detergent composition including alaundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

The cleaning process or the textile care process may for example be alaundry process, a dishwashing process or cleaning of hard surfaces suchas bathroom tiles, floors, table tops, drains, sinks and washbasins.Laundry processes can for example be household laundering, but it mayalso be industrial laundering. Furthermore, the invention relates to aprocess for laundering of fabrics and/or garments where the processcomprises treating fabrics with a washing solution containing a high pHcleaning composition according to the invention, and at least oneprotease variant of the invention. The cleaning process or a textilecare process can for example be carried out in a machine washingprocess, in a manual washing process or in a pre-spotter process. Thewashing solution can for example be an aqueous washing solutioncontaining a cleaning composition.

The invention further concerns the use of cleaning compositions of theinvention in a proteinaceous stain removing processes. The proteinaceousstains may be stains such as food stains, e.g., baby food, sebum, cocoa,egg, blood, milk, ink, grass, or a combination hereof.

A preferred embodiment concerns a method of cleaning, the methodcomprising the steps of: contacting an object with a high pH cleaningcomposition comprising a protease variant of the invention underconditions suitable for cleaning the object. In a preferred embodimentthe cleaning composition is used in a laundry or a dish wash process.

Still another embodiment relates to a method for removing stains fromfabric or dishware which comprises contacting the fabric or dishwarewith a high pH cleaning composition comprising a protease of theinvention under conditions suitable for cleaning the object.

Also contemplated are compositions and methods of treating fabrics(e.g., to desize a textile) using the cleaning composition of theinvention. The high pH cleaning composition can be used in anyfabric-treating method which is well known in the art (see, e.g., U.S.Pat. No. 6,077,316). For example, in one aspect, the feel and appearanceof a fabric is improved by a method comprising contacting the fabricwith a protease in a solution. In one aspect, the fabric is treated withthe solution under pressure.

The high pH cleaning composition of the present invention is suited foruse in liquid laundry and liquid hard surface applications, includingdish wash and car wash. Accordingly, the present invention includes amethod for laundering a fabric or washing a hard surface. The methodcomprises the steps of contacting the fabric/dishware to be cleaned witha solution comprising the high pH cleaning composition according to theinvention. The fabric may comprise any fabric capable of being launderedin normal consumer use conditions. The hard surface may comprise anydishware such as crockery, cutlery, ceramics, plastics such as melamine,metals, china, glass, acrylics or other hard surfaces such as cars,floors etc. The solution preferably has a pH from about 9 to about 13.5.The compositions may be employed at concentrations from about 100 ppm,preferably 500 ppm to about 15,000 ppm in solution. The watertemperatures typically range from about 5° C. to about 95° C., includingabout 10° C., about 15° C., about 20° C., about 25° C., about 30° C.,about 35° C., about 40° C., about 45° C., about 50° C., about 55° C.,about 60° C., about 65° C., about 70° C., about 75° C., about 80° C.,about 85° C. and about 90° C. The water to fabric ratio is typicallyfrom about 1:1 to about 30:1.

In some preferred embodiments, the high pH cleaning compositionsprovided herein are typically formulated such that, during use inaqueous cleaning operations, the wash water has a pH of from about 9 toabout 13.5, or in alternative embodiments, or from about 10 to about13.5 even from about 11 to about 13.5. In some preferred embodiments theliquid laundry products are formulated to have a pH from about 12 toabout 13.5. Techniques for controlling pH at recommended usage levelsinclude the use of buffers, acids, alkalis, etc., and are well known tothose skilled in the art. In the context of the present inventionalkalis are used to adjust pH to about 9 to 13.5 preferably about 10 to13.5.

The Invention is Further Summarized in the Below Paragraphs:

1. A liquid cleaning composition having pH 10 or above, comprising:

(a) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 10

(b) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor.

2. A liquid cleaning composition comprising:

(a) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water;

(b) at least 0.01 wt % protease, wherein the protease is a variant of aparent protease and wherein the protease variant has an amino acidsequence which has at least 60% sequence identity to the amino acidsequence of SEQ ID NO: 2 and wherein said protease variant ischaracterized by having at least 10% higher residual activity than theparent protease when measured after 4 hours at 40° C. in liquiddetergent with pH 7.5 or above, preferably 10;

(c) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor.

3. The liquid cleaning composition of paragraph 1, wherein said pH isfrom about 12 to 13.5.

4. The liquid cleaning composition of any of paragraphs 1-3, wherein theprotease is a variant of a protease having at least 60% identity withthe amino acid sequence of SEQ ID NO: 2 wherein said variant comprises asubstitution at one or more positions selected from the positionscorresponding to the positions 3, 9, 18, 43, 49, 61, 76, 115, 120, 182,185, 188, 194, 205, 206, 209, 216, 217, 218, 222, 255, 256, 259, 260,261 and 262 of BPN′ (SEQ ID NO: 1).

5. The liquid cleaning composition of any of paragraphs 1-4, wherein theprotease is a variant of a protease having at least 60% identity withthe amino acid sequence of SEQ ID NO: 2 wherein said variant comprisesone more substitution corresponding to the substitutions X3V, X9D, X9E,X18S, X43R, X43K, X49T, X61D, X76D, X115W, X120T, X120V, X120D, X182D,X182E, X185E, X185D, X188E, X188D, X194P, X205I, X206L, X209W, X216V,X217M, X218T, X222S, X255E, X255D, X256D, X256E, X259D, X259E, X260A,X260E, X260D, X261M, X261W, X262E and X262D of BPN′ (SEQ ID NO: 1).

6. The liquid cleaning composition of any of paragraphs 1-5, wherein thevariant comprises one more substitution corresponding to thesubstitutions X209W, X262E, X76D, X194P, X204D, X206L.

7. The liquid cleaning composition according to any of the precedingparagraphs, wherein the variant has an amino acid sequence which is atleast 60% identical to SEQ ID NO: 2, e.g., at least 60%, such as atleast 70%, such as at least 80%, such as at least 90%, such as at least95% sequence identity to the amino acid sequence of SEQ ID NO: 2.

8. The liquid cleaning composition according to any of the precedingparagraphs, wherein said protease variant has storage stability at 40°C. greater than at least one stabilized protease variant selected fromthe group consisting of:

S9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E;

S9E+N18S+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+Q206L+Y209W+S259D+N261W+L262ES9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E;

S9E+N76D+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

S9E+N43R+N76D+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N43R+I72A+N76D+A194P+Q206L+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+L262E.

9. The liquid cleaning composition according to any of the precedingparagraphs, wherein said protease variant is selected from the groupconsisting of:

S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E.

10. A liquid cleaning composition comprising:

(a) from 0% to 20% wt, preferably 0.1% to 2.5% wt of a detergentsurfactant;

(b) from 0.001% to 10% wt, preferably 0.1% to 5% wt of a proteasevariant; and

(c1) from 20% to 95% wt, preferably from 40% to 95% wt, furtherpreferably from about 70% to about 90% wt water; and/or

(c2) from 1% to 30% wt of an alkaline buffer system comprising an alkalimetal silicate or an alkali metal hydroxide or a mixture thereof, toprovide a pH of from about 7.5 to 13.5, preferably the pH is from about12 to 13.5;

(d) optionally, a protease inhibitor, preferably said protease inhibitoris a peptide aldehyde protease inhibitor.

11. The liquid cleaning composition of paragraph 10, wherein theprotease variant comprises a substitution at one or more positionsselected from the positions corresponding to the positions 3, 9, 18, 43,49, 61, 76, 115, 120, 182, 185, 188, 194, 205, 206, 209, 216, 217, 218,222, 255, 256, 259, 260, 261 and 262 of BPN′ (SEQ ID NO: 1).

12. The liquid cleaning composition according to paragraph 11, whereinthe protease variant comprises one more substitution corresponding tothe substitutions X3V, X9D, X9E, X18S, X43R, X43K, X49T, X61D, X76D,X115W, X120T, X120V, X120D, X182D, X182E, X185E, X185D, X188E, X188D,X194P, X205I, X206L, X209W, X216V, X217M, X218T, X222S, X255E, X255D,X256D, X256E, X259D, X259E, X260A, X260E, X260D, X261M, X261W, X262E andX262D of BPN′ (SEQ ID NO: 1).

13. The liquid cleaning composition according to any of paragraphs 10 to12, wherein the protease variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% but less than 100%sequence identity to the polypeptide of SEQ ID NO: 2.

14. The liquid cleaning composition according to any of paragraphs10-13, wherein said protease variant is selected from the groupconsisting of:

S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E;

S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH;

S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E;

S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S3V+N76D+H120V+Q182E+N185E+S188E+V205I+Q206L+Y209W+S216V+S256D+N261W+L262E;

S9E+N43R+N76D+A158E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E;S9E+N43R+N76D+A158E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S216V+L262E;S9E+N43R+N76D+A158E+G160P+S161E+A194P+Q206L+Y209W+S259D+L262E+*275aH+*275bH;S9E+N43R+N76D+A158E+G160P+S161E+A194P+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+A158E+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A158E+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+S256D+S259D+N261W+L262E;S9E+N43R+N76D+A194P+Q206L+Y209W+T255E+S256D+S259D+T260E+N261W+L262E;S9E+N43R+N76D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G115W+H120V+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G115W+H120V+P129D+A194P+Q206L+Y209W+S259D+L262E;S9E+N43R+N76D+G160P+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E;S9E+N43R+N76D+G160P+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+G160P+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S216V+S259D+N261W+L262E;S9E+N43R+N76D+S161E+V205I+Q206L+Y209W+S259D+N261W+L262E;S9E+N43R+N76D+P131*+A194P+Q206L+Y209W+S259D+L262E;

S9E+N43R+N76D+H120V+Q182E+A194P+V205I+Q206L+Y209W+S256D+N261W+L262E+*275aH+*275bH;S9E+N43R+N76D+S161E+A194P+N204D+V205I+Q206L+Y209W+S212G+S216V+L262E+*275aH+*275bH;

S9E+N43R+N76D+V205I+Q206L+Y209W+S212G+S259D+N261W+L262E;S9E+N43R+N76D+N204D+V205I+Q206L+Y209W+S212G+S216V+S259D+N261W+L262E.

15. The cleaning composition according to any of the precedingparagraphs, which comprises one or more additional enzymes selected fromthe group comprising of amylases, catalases, cellulases (e.g.,endoglucanases), cutinases, haloperoxygenases, lipases, mannanases,pectinases, pectin lyases, peroxidases, proteases, xanthanases, andxyloglucanases, or any mixture thereof.

16. Use of the liquid cleaning composition of any of the precedingparagraphs in a cleaning process such as laundry or hard surfacecleaning including dish wash and industrial cleaning.

17. The use of paragraph 16, wherein the laundry process is selectedfrom a group consisting of: a residential laundry process, an industriallaundry process and an institutional laundry process.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials and Methods Suc-AAPF-pNA Activity Assay

Proteolytic activity can be determined by a method employingSuc-AAPF-PNA as the substrate. Suc-AAPF-PNA is an abbreviation forN-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide, and isa blocked peptide which can be cleaved by endo-proteases. Followingcleavage a free PNA molecule is liberated which has a yellow color andthus can be measured by visible spectrophotometry at wavelength 405 nm.The Suc-AAPF-PNA substrate is manufactured by Bachem (cat. no. L1400,dissolved in DMSO).

The protease sample to be analyzed is diluted in residual activitybuffer (100 mM Tris pH 8.6). The assay is performed by transferring 3 0μl of diluted enzyme samples to 96 well microtiter plate and adding 70μl substrate working solution (0.72 mg/ml in 100 mM Tris pH8.6). Thesolution was mixed at room temperature and absorption is measured every20 seconds over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curveis directly proportional to the activity of the protease in questionunder the given set of conditions. The protease sample is diluted to alevel where the slope was linear.

Example 1: Preparation and Expression of Protease Variants

The following summarizes the mutation and introduction of an expressioncassette into Bacillus subtilis. All DNA manipulations were done by PCR(e.g., Sambrook et al.; Molecular Cloning; Cold Spring Harbor LaboratoryPress).

Recombinant B. subtilis constructs encoding protease variants were usedto inoculate shakeflasks containing a rich media (e.g., 100 g/L sucrose(Danisco cat. no. 109-0429), 40 g/L crust soy (soy bean flour), 10 g/LNa₂HPO₄.12H₂O (Merck cat. no. 6579), 0.1 ml/L replace-Dowfax63N10 (Dow).Cultivation typically takes 4 days at 30° C. shaking with 220 rpm.

Example 2: Purification of Protease Variants

The culture broth was centrifuged at 26000×g for 20 minutes and thesupernatant was carefully decanted from the precipitate. The supernatantwas filtered through a Nalgene 0.2 μm filtration unit in order to removethe rest of the Bacillus host cells. The pH in the 0.2 μm filtrate wasadjusted to pH 8 with 3 M Tris base and the pH adjusted filtrate wasapplied to a MEP Hypercel column (Pall Corporation) equilibrated in 20mM Tris/HCl, 1 mM CaC₂, pH 8.0. After washing the column with theequilibration buffer, the column was step-eluted with 20 mMCH₃COOH/NaOH, 1 mM CaCl₂, pH 4.5. Fractions from the column wereanalyzed for protease activity using the Suc-AAPF-pNA assay at pH 9 andpeak-fractions were pooled. The pH of the pool from the MEP Hypercelcolumn was adjusted to pH 6 with 20% (v/v) CH₃COOH or 3 M Tris base andthe pH adjusted pool was diluted with deionized water to the sameconductivity as 20 mM MES/NaOH, 2 mM CaCl₂, pH 6.0. The diluted pool wasapplied to a SP-Sepharose® Fast Flow column (GE Healthcare) equilibratedin 20 mM MES/NaOH, 2 mM CaC₂, pH 6.0. After washing the column with theequilibration buffer, the protease variant was eluted with a linear NaClgradient (0-->0.5 M) in the same buffer over five column volumes.Fractions from the column were analyzed for protease activity using theSuc-AAPF-pNA assay at pH 9 and active fractions were analyzed bySDS-PAGE. Fractions, where only one band was observed on the Coomassiestained SDS-PAGE gel, were pooled as the purified preparation and wasused for further experiments.

Example 3: Accelerated Storage Stability Assay

Storage stability of protease variants in liquid detergent was evaluatedusing an accelerated assay with incubation at elevated temperatures forup to 24 hours.

All purified protease samples were diluted to concentrations of 0.2 and0.1 mg/ml based on absorbance at 280 nm and theoretical extinctioncoefficient using 0.01% Triton X-100. For each variant 2 wells with highprotease concentration and 2 wells with low concentration were included.As reference Savinase (SEQ ID NO 2) or a stabilized protease variant wasincluded on each microtiter plate. 30 μl protease sample was mixed with270 μl detergent (Surf Sparkling Ocean, DC-2014-0009) in the well of amicrotiter plate (Nunc U96 PP 0.5 ml) using a magnetic bar (on Zephyrpipetting station (Caliper LifeSciences) for 30 min). 20 μl of thismixture was then transferred to another microtiter plate (Nunc U96 PP0.5 ml with added magnetic bars) and mixed with 150 μl 100 mM Tris pH8.6 (at least 5 min on Zephyr). 30 μl of this dilution was transferredto a Nunc F 96-MTP, and after addition of 70 μl substrate solutioninitial activity of unstressed sample was determined by measuringabsorbance at 405 nm every 20 sec for 5 min (on a SpectraMax Plus).After sealing, the detergent plate was incubated at 50° C. and pH 11 inan Eppendorf Thermomixer (no shaking). After 1, 3-4 and 23-24 hoursincubation, 20 μl samples were withdrawn and residual activity ofstressed sample was measured as with the initial, unstressed activity.

Decrease in activity during incubation with detergent was assumed to beexponential. Half lifes (T½) were found from linear regression ofLog(Activity) versus incubation time. Savinase (SEQ ID NO 2) has a veryshort half life under the conditions of this experiment and thereforethe effects of the mutations were measured relative to an alreadystabilized protease variant.

Accelerated Storage Stability at 40° C. of Variants T½ IF: Half LifeImprovement Effect of Single Mutations Mutation G61ES9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E, T½: 0.9S9E+N43R+G61E+N76D+G115W+H120V+A194P+Q206L+S259D+L262E, T½: 4.8 MutationY209W S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E, T½: 4.5S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E, T½: 13.0S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E, T½: 4.6S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E, T½: 12.4Mutation M222S S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E, T½:12.0 S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+M222S+L262E, T½: 14.5Mutation V205IS9E+N43R+N76D+A194P+Q206L+T255E+S256D+S259D+T260E+N261W+L262E, T½: 5.0S9E+N43R+N76D+A194P+V205I+Q206L+T255E+S256D+S259D+T260E+N261W+L262E, T½:1.2 S9E+N43R+N76D+N117H+H120D+A194P+Q206L+S259D+L262E, T½: 5.0S9E+N43R+N76D+N117H+H120D+A194P+V205I+Q206L+S259D+L262E, T½: 1.6S9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E, T½: 3.0S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E, T½: 6.6S9E+N18S+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E, T½: 1.0S9E+N18S+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E, T½: 4.6S9E+N43R+N76D+G115W+H120V+A194P+Q206L+S259D+L262E, T½: 0.9S9E+N43R+N76D+G115W+H120V+A194P+V205I+Q206L+S259D+L262E, T½: 5.0S9E+N43R+N76D+A194P+Q206L+Y209W+S259D+N261W+L262E, T½: 9.9S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E, T½: 13.0Mutation S256D S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E, T½:4.5 S9E+N43R+N76D+A194P+V205I+Q206L+S256D+S259D+N261W+L262E, T½: 6.6S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S259D+N261W+L262E, T½: 12.4S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+Y209W+S256D+S259D+N261W+L262E, T½:13.3 Mutation S188E S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E,T½: 12.0 S9E+N43R+N76D+S188E+A194P+V205I+Q206L+Y209W+S216V+L262E, T½:13.7 Mutation T260ES9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E, T½: 13.5S9E+N76D+G115W+G160P+Q182E+V205I+Q206L+Y209W+S256D+T260E+N261W+L262E,T½: 15.1 Mutation H120TS9E+N43R+N76D+A194P+Q206L+S256D+S259D+N261W+L262E, T½: 3.0S9E+N43R+N76D+H120T+A194P+Q206L+S256D+S259D+N261W+L262E, T½: 5.4S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E, T½: 12.0S9E+N43R+N76D+H120T+A194P+V205I+Q206L+Y209W+S216V+L262E, T½: 12.2Mutation G160P S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E, T½:13.2 S9E+N76D+G160P+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E, T½: 14.3Mutation Q182E S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E, T½:4.5 S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E, T½: 4.6S9E+N76D+V205I+Q206L+Y209W+S256D+N261W+L262E, T½: 12.3S9E+N76D+Q182E+V205I+Q206L+Y209W+S256D+N261W+L262E, T½: 13.2S9E+N43R+N76D+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH, T½: 4.2S9E+N43R+N76D+Q182E+A194P+V205I+Q206L+S259D+N261W+L262E+*275aH, T½: 4.6S9E+N43R+N76D+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E, T½: 11.5S9E+N43R+N76D+Q182E+N185E+S188E+A194P+Q206L+Y209W+S259D+L262E, T½: 14.0Mutation N185E S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E, T½:12.0 S9E+N43R+N76D+N185E+A194P+V205I+Q206L+Y209W+S216V+L262E, T½: 12.4Mutation N261M S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+L262E, T½:12.0 S9E+N43R+N76D+A194P+V205I+Q206L+Y209W+S216V+N261M+L262E, T½: 12.7Mutation S259D S9E+N43R+I72A+N76D+A194P+Q206L+L262E, T½: 6.0S9E+N43R+I72A+N76D+A194P+Q206L+S259D+L262E, T½: 7.2S9E+N43R+N76D+A194P+V205I+Q206L+L262E, T½: 6.0S9E+N43R+N76D+A194P+V205I+Q206L+S259D+L262E, T½: 6.5 Example 4: FullScale Wash Results for the Proteases of the Invention

The wash performance of the proteases of the invention was tested infull scale wash. The wash performance was tested on 22 different stainsat a dosage response in laundry liquid detergent.

After washing and rinsing the swatches were spread out flat and allowedto air dry at room temperature overnight. All washes were evaluated theday after the wash. Light reflectance evaluations of the swatches weredone using a Macbeth Color Eye 7000 reflectance spectrophotometer withvery small aperture. The measurements were made without UV in theincident light and remission at 460 nm was extracted. Measurements weremade on unwashed and washed swatches. The test swatch to be measured wasplaced on top of another swatch of same type and color.

Calculating the enzyme effect was done by taking the measurements fromwashed swatches with enzymes and subtract with the measurements fromwashed without enzyme for each stain, ΔRem_(enzyme). Wash performance isexpressed as a delta remission value (ΔRem).

The experiments were conducted with the detergent composition andswatches under the experimental conditions as specified in Table 21below.

TABLE 21 Experimental conditions for full scale wash laundry experimentsDetergent dosage Purex (Henkel) 45 g/wash Arm & Hammer (C&D) 45 g/washXtra (C&D) 45 g/wash Triple Clean (SUN) 45 g/wash ALL Free & Clear (SUN)45 g/wash Test solution volume 63 L pH As is Wash time 12 min main wash,2 rinses Temperature 30/15° C. Water hardness 6.7° dH Proteaseconcentration 0.25, 0.5, 1 wt % SwatchesWater hardness was adjusted to 6.7° dH by addition of CaCl₂), MgCl2, andNaHCO3 (Ca2+:Mg2+:CO3-=3:1:6) to the test system.

TABLE 22 Delta remission value of detergent comprising proteasescompared to detergent without protease at 15° C. in all Free & Clear(The enzymes tested were the following: 1: S9E + N43R + N76D + V205I +Q206L + Y209W + S259D + N261W + L262E; 2: S9E + N43R + N76D + H120V +Q182E + A194P + V205I + Q206L + Y209W + S256D + N261W + L262E + *275aH +*275bH; 3: S3V + N76D + H120V + Q182E + N185E + S188E + V205I + Q206L +Y209W + S216V + S256D + N261W + L262E): Delta (vs blank) Enzyme dose %0.25% 0.50% 1.00% Enzymes 1 2 3 1 2 3 1 2 3 108KC ACD 0.3 0.9 0.5 1.30.3 1.3 1.1 1.6 0.8 sheep blood - dbl app Blood, Hard 0.7 0.3 0.8 1.61.7 1.0 1.4 2.2 1.1 set Blood, Soft set −0.7 2.0 −1.7 −0.8 −0.1 2.5 1.2−0.8 0.9 (Cotton/Polyester) CS-01 Aged −0.9 −0.1 −0.2 −1.0 −0.1 −0.6 0.6−0.3 −0.3 blood EMPA111 −0.3 0.3 −0.5 −0.4 −0.6 −0.3 −0.3 0.4 −0.2 Blood062KC 0.7 1.5 0.7 1.3 0.1 1.2 0.7 2.5 1.0 Rubbed in grass 062PE 1.2 0.4−0.2 2.0 −0.5 0.2 0.7 1.5 0.7 Rubbed in grass C-H039 0.5 0.9 0.9 0.8−0.2 0.5 0.6 1.3 0.4 Squeezed Grass CS-08 Grass −0.2 −0.2 0.1 −0.1 0.10.0 0.1 −0.4 0.3 Extract EMPA164 0.6 0.5 −0.2 0.8 0.7 −0.2 0.9 0.4 0.6Grass Grass, cotton −0.1 0.6 0.0 0.3 0.0 0.7 0.7 1.4 0.9 NZ Grass CN 0.90.6 −0.6 −0.3 1.1 0.9 1.0 0.8 1.3 Grass PCS-8 Grass 0.2 0.1 −0.1 −0.2−0.1 0.0 0.0 0.0 0.2 Extract C-05 BMI 0.0 0.6 −0.3 0.9 0.3 1.1 0.6 1.21.6 EMPA 116 0.4 −0.6 0.4 1.7 1.3 1.3 2.8 2.4 1.7 BMI © EMPA 117 0.4 1.2−0.2 2.3 0.9 1.8 3.7 3.1 3.1 BMI (P/C) 038KC Choc. 0.5 0.0 0.4 0.5 −0.40.9 0.0 0.4 1.1 Milk PC-03 Choc. 0.6 0.2 0.0 1.1 0.4 0.5 1.5 1.1 1.1Milk/carbon black 051KC Egg 0.0 −0.1 0.2 −0.3 0.0 0.0 0.5 0.0 0.0 W10NWhole 1.4 0.9 1.0 1.8 1.6 1.5 2.2 2.1 2.0 Egg French's 0.7 0.6 0.3 1.10.7 0.4 0.9 0.7 0.6 brown gravy (contains wheat protein, beef extract)KC-H172 0.8 2.2 0.3 2.1 0.8 2.1 1.9 2.2 0.8 Meat pate

TABLE 23 Delta remission value of detergent comprising proteasescompared to detergent without protease at 30° C. in all Free & Clear(The enzymes tested in “Results(Enzymes-Blank)” below were thefollowing: 1: S9E + N43R + N76D + V205I + Q206L + Y209W + S259D +N261W + L262E; 2: S9E + N43R + N76D + Q182E + A194P + V205I + Q206L +Y209W + S256D + S259D + N261W + L262E; 3: S9E + N43R + N76D + H120V +Q182E + A194P + V205I + Q206L + Y209W + S256D + N261W + L262E + *275aH +*275bH; 4: S3V + N76D + H120V + Q182E + N185E + S188E + V205I + Q206L +Y209W + S216V + S256D + N261W + L262E): All free and clear 30° C. Ref:Detergent blank Results (Enzymes- 0.50% 1% Blank) 1 2 3 4 1 2 3 4 Blood108KC ACD 3.3 2.8 2.5 2.2 4.5 4.0 4.0 3.2 sheep blood - dbl app Blood,Hard 1.9 1.1 1.4 0.9 3.3 1.8 2.4 1.4 set Blood, Soft 1.6 1.3 2.0 0.9 1.92.1 2.4 1.8 set (Cotton/Polyester) CS-01 Aged 1.7 2.2 2.8 2.0 3.5 3.23.7 2.3 blood EMPA111 1.4 2.2 1.9 1.9 2.8 2.9 3.2 2.7 Blood Grass 062KC2.3 2.5 1.9 1.5 4.2 3.3 2.6 3.2 Rubbed in grass C-H039 0.4 0.3 0.8 0.00.2 0.3 0.8 0.5 Squeezed Grass CS-08 Grass 0.2 0.0 −0.1 −0.1 0.4 0.1 0.20.2 Extract PCS-8 Grass 0.3 0.3 0.7 0.2 0.9 0.5 0.5 0.7 Extract EMPA1641.3 1.0 0.9 0.8 1.8 0.6 1.3 0.8 Grass Grass on 2.7 1.8 3.1 1.8 3.7 3.13.0 2.4 cotton NZ grass 3.6 4.5 3.6 3.4 5.5 4.1 5.0 4.0 BMI C-05 BMI 3.73.7 4.1 3.4 7.2 6.6 6.9 5.4 EMPA 116 6.4 7.0 6.3 5.9 8.2 8.5 7.7 7.7 BMI(C) EMPA 117 9.3 9.6 9.2 8.0 12.7 12.8 13.3 11.5 BMI (P/C) Cocoa 038KCChoc. 1.5 1.5 1.9 1.8 2.6 2.3 1.4 1.6 Milk PC-03 Choc. 3.4 2.9 3.0 2.74.7 4.8 4.3 3.9 Milk/carbon black Egg 051KC Egg 2.9 2.9 2.9 2.5 3.2 3.03.0 2.9 10N Whole 4.0 4.2 4.4 4.1 5.5 5.3 5.4 5.3 Egg Food KC-H172 3.83.0 4.2 3.6 5.1 5.1 6.3 5.4 stain Meat pate French's 1.4 1.2 1.5 0.9 1.51.3 1.5 1.3 brown gravy

TABLE 24 Delta remission value of detergent comprising proteasescompared to detergent without protease at 30° C. in all Arm & Hammer(The enzymes tested were the following: 1: S9E + N43R + N76D + V205I +Q206L + Y209W + S259D + N261W + L262E; 2: S9E + N43R + N76D + H120V +Q182E + A194P + V205I + Q206L + Y209W + S256D + N261W + L262E + *275aH +*275bH; 3: S3V + N76D + H120V + Q182E + N185E + S188E + V205I + Q206L +Y209W + S216V + S256D + N261W + L262E): Delta (vs blank) Detergent Arm &Hammer Enzyme dose % 0.25% 0.50% 1.00% Enzymes 1 2 3 1 2 3 1 2 3 108KC0.09 0.84 0.92 1.70 0.81 0.64 1.57 2.47 1.39 ACD sheep blood - dbl appBlood, 1.74 1.12 0.82 2.42 1.50 0.86 2.57 3.34 1.45 Hard set Blood, Soft5.18 3.54 3.78 6.79 7.91 6.99 6.96 1.01 3.04 set (Cotton/Polyester)CS-01 0.84 0.20 −0.18 0.25 0.20 −0.14 2.16 −0.03 0.38 Aged blood EMPA1110.27 0.60 −0.16 0.74 0.88 0.24 2.05 1.35 1.21 Blood 062KC 1.03 2.81 0.401.23 −0.18 1.08 2.94 2.73 0.24 Rubbed in grass 062PE 2.45 1.14 0.64 1.622.88 1.88 2.25 1.75 0.53 Rubbed in grass C-H039 0.78 1.35 0.65 0.32 1.011.29 0.91 0.92 0.25 Squeezed Grass CS-08 0.11 0.16 0.49 0.31 0.23 0.370.20 0.18 0.15 Grass Extract EMPA164 0.27 0.24 0.11 0.65 −0.28 0.20 0.550.85 −0.18 Grass Grass, 0.52 0.73 0.12 0.94 0.89 0.35 0.99 1.25 0.75cotton NZ Grass 2.12 1.33 0.31 1.91 1.50 1.31 2.41 2.88 1.31 CN GrassPCS-8 0.34 0.03 0.27 0.58 0.39 0.13 0.59 0.32 0.40 Grass Extract C-05BMI 0.96 1.47 −0.78 0.79 0.97 0.29 2.83 2.53 1.85 EMPA 116 3.54 2.671.93 5.35 4.24 3.45 6.71 6.27 4.81 BMI © EMPA 117 5.03 4.31 3.95 8.116.62 6.10 9.96 8.48 6.74 BMI (P/C) 038KC 2.69 2.43 2.94 3.36 2.24 4.283.22 3.43 3.70 Choc. Milk PC-03 1.77 2.04 1.26 2.66 2.28 1.67 3.79 3.132.57 Choc. Milk/carbon black 051KC 0.96 0.84 0.63 2.08 1.38 1.10 2.542.32 2.28 Egg W10N 2.05 2.31 2.12 3.23 3.25 3.11 4.14 3.71 3.62 WholeEgg French's 0.83 0.87 0.79 0.86 0.90 0.65 0.97 1.06 0.80 brown gravy(contains wheat protein, beef extract) KC-H172 3.48 3.46 3.43 4.23 4.363.64 5.65 5.66 3.85 Meat pate

TABLE 25 Delta remission value of detergent comprising proteasescompared to detergent without protease at 30° C. in Purex (The enzymestested were the following: 1: S9E + N43R + N76D + V205I + Q206L +Y209W + S259D + N261W + L262E; 2: S9E + N43R + N76D + H120V + Q182E +A194P + V205I + Q206L + Y209W + S256D + N261W + L262E + *275aH + *275bH;3: S3V + N76D + H120V + Q182E + N185E + S188E + V205I + Q206L + Y209W +S216V + S256D + N261W + L262E): Purex Enzyme dose % 0.25% 0.50% 1.00%Enzymes 1 2 3 1 2 3 1 2 3 108KC 0.55 0.42 0.51 1.55 1.33 0.68 1.79 1.361.42 ACD sheep blood - dbl app Blood, 0.51 0.17 0.30 0.77 0.45 0.39 1.500.99 0.56 Hard set Blood, Soft −2.95 −1.96 −2.89 3.62 5.20 −2.08 6.985.58 −1.45 set (Cotton/Polyester) CS-01 0.54 −0.58 −0.30 1.02 −0.08−0.39 1.96 1.84 0.48 Aged blood EMPA111 0.11 0.43 0.26 0.51 1.00 −0.621.16 1.07 1.37 Blood 062KC 1.82 1.77 2.05 1.77 1.37 1.04 1.78 2.22 1.73Rubbed in grass 062PE 3.31 3.69 2.40 4.40 3.25 3.66 5.56 5.31 3.98Rubbed in grass C-H039 −0.44 0.81 0.09 0.48 0.29 0.06 0.40 0.89 −0.09Squeezed Grass CS-08 −0.55 −0.15 −0.28 −0.14 −0.05 −0.16 −0.13 −0.89−0.10 Grass Extract EMPA164 1.16 0.98 0.75 1.24 1.44 0.98 1.12 1.48 0.66Grass Grass, 0.91 1.18 1.39 1.37 1.34 1.08 2.18 2.22 1.67 cotton NZGrass 1.61 1.85 1.68 2.77 2.12 1.19 3.55 3.32 1.86 CN Grass PCS-8 0.670.60 0.47 1.07 0.46 0.21 0.82 0.58 0.49 Grass Extract C-05 BMI 0.65−0.33 0.37 2.33 1.44 0.12 3.94 2.38 2.32 EMPA 116 3.02 3.10 3.46 5.105.12 3.19 7.53 6.87 4.95 BMI © EMPA 117 3.75 4.98 4.87 6.94 5.97 5.609.59 8.81 8.43 BMI (P/C) 038KC −0.22 −0.12 1.93 1.23 1.64 1.21 1.59 1.921.07 Choc. Milk PC-03 0.98 1.17 1.26 2.20 2.07 1.46 3.75 3.34 2.47 Choc.Milk/carbon black 051KC 0.74 0.33 0.59 1.78 1.17 1.04 2.29 2.27 1.99 EggW10N 1.91 1.85 1.97 2.88 2.65 2.33 3.81 3.48 3.33 Whole Egg French's0.70 0.52 0.14 0.87 0.64 0.44 0.82 0.62 0.73 brown gravy (contains wheatprotein, beef extract) KC-H172 2.80 3.27 3.31 4.16 4.56 4.52 5.37 6.305.41 Meat pate

TABLE 26 Delta remission value of detergent comprising proteasescompared to detergent without protease at 30° C. in Sun Triple Clean(The enzymes tested were the following: 1: S9E + N43R + N76D + V205I +Q206L + Y209W + S259D + N261W + L262E; 2: S9E + N43R + N76D + H120V +Q182E + A194P + V205I + Q206L + Y209W + S256D + N261W + L262E + *275aH +*275bH; 3: S3V + N76D + H120V + Q182E + N185E + S188E + V205I + Q206L +Y209W + S216V + S256D + N261W + L262E): Delta (vs blank) Detergent SunTriple Clean Enzyme dose % 0.25% 0.50% 1.00% Enzymes 1 2 3 1 2 3 1 2 3108KC ACD 1.94 1.03 0.20 1.67 2.12 1.57 2.19 2.65 1.54 sheep blood - dblapp Blood, Hard set −0.43 −0.73 −1.39 0.06 −0.20 −0.83 0.63 −0.11 −0.65Blood, Soft set 2.48 0.77 0.29 2.65 3.21 2.79 5.52 2.33 2.16(Cotton/Polyester) CS-01 Aged −0.02 −0.11 −0.52 0.92 0.28 −0.29 0.881.55 0.03 blood EMPA111 0.57 0.40 −0.29 1.29 0.78 0.69 1.56 1.65 0.73Blood 062KC Rubbed 1.13 1.58 0.06 1.89 1.90 0.50 2.48 2.61 1.12 in grass062PE Rubbed 2.43 1.78 1.58 2.29 2.46 1.74 3.37 2.61 1.36 in grassC-H039 1.19 1.76 1.36 1.17 1.24 1.39 1.64 1.78 1.07 Squeezed Grass CS-08Grass 0.11 −0.19 −0.33 −0.14 −0.15 −0.13 0.25 0.37 0.00 Extract EMPA1640.17 0.28 0.07 0.50 0.13 0.18 −0.11 0.33 0.27 Grass Grass, cotton 0.850.89 0.79 1.26 1.63 0.81 1.83 1.58 1.36 NZ Grass CN 1.32 1.25 0.85 2.551.72 1.38 2.91 2.93 1.64 Grass PCS-8 Grass 0.06 −0.04 −0.10 0.20 −0.08−0.01 0.11 −0.05 −0.10 Extract C-05 BMI 1.48 0.62 1.18 2.30 2.42 1.414.07 3.26 2.22 EMPA 116 BMI © 4.09 3.18 2.96 5.89 4.27 3.85 6.78 6.055.24 EMPA 117 BMI 4.03 3.97 3.68 6.41 6.35 5.28 8.96 8.25 5.95 (P/C)038KC Choc. 2.48 0.83 1.55 2.24 2.01 3.11 2.63 3.20 2.68 Milk PC-03Choc. 1.63 1.40 1.28 2.18 2.08 1.88 3.77 3.29 2.36 Milk/carbon black051KC Egg 0.35 0.12 0.20 1.14 0.95 0.66 2.14 2.27 1.75 W10N Whole 1.411.31 1.24 2.25 2.16 2.04 3.07 1.20 2.55 Egg French's brown 0.72 0.720.54 0.93 0.63 0.73 1.00 0.90 0.82 gravy (contains wheat protein, beefextract) KC-H172 Meat 2.72 2.62 1.68 3.04 3.95 3.03 4.18 4.69 4.91 pate

TABLE 27 Delta remission value of detergent comprising proteasescompared to detergent without protease at 30° C. in Xtra. The enzymestested included the following: 1: S9E + N43R + N76D + V205I + Q206L +Y209W + S259D + 0 N261W + L262E; 2: S9E + N43R + N76D + H120V + Q182E +A194P + V205I + Q206L + Y209W + S256D + N261W + L262E + *275aH + *275bH;3: S3V + N76D + H120V + Q182E + N185E + S188E + V205I + Q206L + Y209W +S216V + S256D + N261W + L262E: Delta (vs blank) Detergent Xtra Enzymedose % 0.25% 0.50% 1.00% Enzymes 1 2 3 1 2 3 1 2 3 108KC ACD 1.56 1.160.96 2.34 2.07 1.48 2.74 3.03 2.29 sheep blood - dbl app Blood, Hard set0.34 0.24 −0.31 0.97 0.08 −0.13 1.31 1.40 0.35 Blood, Soft set 5.55 3.964.28 6.05 3.89 0.93 5.47 4.80 2.77 (Cotton/Polyester) CS-01 Aged 0.320.71 0.66 1.96 1.66 1.04 2.82 2.25 1.88 blood EMPA111 Blood 0.78 0.460.42 1.53 1.50 1.17 1.78 2.51 1.73 062KC Rubbed in 0.00 1.03 1.30 2.001.29 0.85 1.86 2.81 1.59 grass 062PE Rubbed in 2.74 2.39 2.46 5.43 2.102.41 2.83 5.13 3.53 grass C-H039 0.49 0.08 0.80 0.60 0.00 0.74 1.23 1.340.38 Squeezed Grass CS-08 Grass 0.13 0.24 −0.12 0.35 0.11 −0.49 0.300.38 0.73 Extract EMPA164 Grass 0.37 0.51 0.08 0.82 0.50 −0.16 0.71 1.080.44 Grass, cotton 1.40 1.11 0.06 1.74 1.03 0.63 1.44 1.97 1.03 NZ GrassCN 1.43 1.43 1.05 2.42 1.61 1.22 3.24 3.19 2.25 Grass PCS-8 Grass 1.060.99 0.64 0.81 0.66 −0.11 0.90 0.98 0.73 Extract C-05 BMI 1.58 0.92 0.932.32 1.65 0.35 3.00 2.31 1.98 EMPA 116 BMI © 3.33 3.12 2.10 4.86 4.553.62 6.97 7.02 5.47 EMPA 117 BMI 4.04 3.94 3.37 7.11 5.29 4.31 8.36 8.756.84 (P/C) 038KC Choc. Milk 0.91 1.09 1.64 2.85 1.50 1.89 2.89 2.72 2.10PC-03 Choc. 1.73 1.26 1.10 2.45 1.81 1.47 3.19 3.75 2.89 Milk/carbonblack 051KC Egg 0.91 0.59 0.45 1.90 1.37 1.03 2.49 2.89 2.32 W10N WholeEgg 2.42 2.16 2.09 3.22 2.98 2.78 4.00 3.79 3.82 French's brown 0.980.97 0.68 0.97 0.73 0.58 1.12 1.08 0.93 gravy (contains wheat protein,beef extract) KC-H172 Meat 1.12 2.20 0.83 3.75 3.18 2.40 3.96 4.68 4.16pate

Example 5: Test of Subtilisin 309 Variants in Mini Wash

The wash performance of the proteases of the invention was tested usinglaundry liquid detergent on one technical stain using the mini washsystem.

The Mini wash assay is a test method where soiled textile iscontinuously lifted up and down into the test solution and subsequentlyrinsed.

TABLE 28 The wash experiment is conducted under the experimentalconditions specified below: Detergent Tixan - YPE commercial availabledetergent Detergent dose 2 g/l pH As is (i.e. not adjusted) Waterhardness 8.4° dH. Enzyme conc. Example 20-40-80 nM Test solution 50 mlvolume Test material EMPA117 EH Blood/milk/ink on cotton/polyester extraheated Temperature 25° C. Wash time 20 min soak + 18 min main wash Rinsetime 10 min Test system Soiled textile continuously lifted up and downinto the test solutions, 50 times per minute (up-time 0.29 sec,down-time 0.29 sec, lift time 0.17 sec). The test solutions are kept in125 ml glass beakers. After wash of the textiles are continuously liftedup and down into tap water, 50 times per minute (up-time 0.5 sec,down-time 5 sec, lift time 0.5 sec).

Test materials were obtained from EMPA Testmaterials AG Movenstrasse 12,CH-9015 St. Gallen, Switzerland.

The textiles were subsequently air-dried and the wash performance wasmeasured as the brightness of the color of these textiles. Brightnesscan also be expressed as the Remission (R), which is a measure for thelight reflected or emitted from the test material when illuminated withwhite light. The Remission (R) of the textiles was measured at 460 nmusing a Zeiss MCS 521 VIS spectrophotometer. The measurements were doneaccording to the manufacturer's protocol.

Calculating the enzyme effect was done by taking the measurements fromwashed swatches with enzymes and subtract with the measurements fromwashed without enzyme for each stain, ΔRem_(enzyme).

The experiments were conducted as described in the mini wash assay forlaundry method with the detergent composition and swatches under theexperimental conditions as specified in Table 29 below.

TABLE 29 Experimental conditions for mini wash laundry experimentsDetergent dosage YPE, 2 g/L Test solution volume 50 mL pH As is Washtime 20 min soak + 18 min main wash Temperature 25° C. Water hardness8.4° dH Protease concentration 0-20-40-80 nM Swatch EMPA 117 EH

Water hardness was adjusted to 8.4° dH by addition of CaC₂, MgCl₂, andNaHCO₃ (Ca²⁺:Mg²⁺:CO₃ ⁻=2:1:4.5) to the test system. After washing thetextiles were rinsed in tap water and dried.

TABLE 30 Relative performance of proteases subtilisin 309 variantscompared to detergent with Subtilisin 309 (SEQ ID NO: 2) at 25° C.Enzyme 20 nM 40 nM 80 nM Subtilisin309 (SEQ ID NO: 2) 1.0 1.0 1.0 S9E +N43R + N76D + A158E + A194P + N204D + V205I + 1.1 1.3 1.1 Q206L +Y209W + S212G + S216V + L262E S9E + N43R + N76D + A158E + G160P +V205I + Q206L + 1.1 1.5 1.5 Y209W + S259D + N261W + L262E S9E + N43R +N76D + A158E + G160P + A194P + N204D + 1.1 1.2 1.3 V205I + Q206L +Y209W + S212G + S216V + L262E S9E + N43R + N76D + A158E + G160P +S161E + A194P + 0.9 1.1 1.1 N204D + V205I + Q206L + Y209W + S216V +L262E + *275aH + *275bH S9E + N43R + N76D + A158E + G160P + S161E +A194P + 0.8 1.0 1.1 N204D + V205I + Q206L + Y209W + L262E S9E + N43R +N76D + A158E + G160P + S161E + A194P + 0.8 0.9 1.3 N204D + V205I +Q206L + Y209W + S212G + S216V + L262E + *275aH + *275bH S9E + N43R +N76D + A158E + G160P + S161E + A194P + 1.0 1.2 1.4 N204D + V205I +Q206L + Y209W + S212G + L262E S9E + N43R + N76D + A158E + A194P +V205I + Q206L + 0.9 1.3 1.5 Y209W + S259D + N261W + L262E S9E + N43R +N76D + A158E + G160P + S161E + A194P + 0.7 0.9 1.0 N204D + V205I +Q206L + Y209W + S212G + L262E + *275aH + *275bH S9E + N43R + N76D +A158E + G160P + S161E + A194P + 1.1 1.2 1.3 N204D + V205I + Q206L +Y209W + S216V + L262E S9E + N43R + N76D + A158E + G160P + S161E +A194P + 0.9 0.8 1.1 Q206L + Y209W + S259D + L262E + *275aH + *275bHS9E + N43R + N76D + A158E + G160P + S161E + A194P + 1.2 1.2 1.3 V205I +Q206L + Y209W + S212G + S216V + L262E S9E + N43R + N76D + A158E +S161E + V205I + Q206L + 0.8 1.2 1.4 Y209W + S259D + N261W + L262E S9E +N43R + N76D + A158E + G160P + S161E + V205I + 0.7 1.1 1.1 Q206L +Y209W + S259D + N261W + L262E S9E + N43R + N76D + A158E + S161E +A194P + N204D + 1.0 1.2 1.1 V205I + Q206L + Y209W + S212G + S216V +L262E S9E + N43R + N76D + G160P + V205I + Q206L + Y209W + 1.1 1.3 1.3S259D + N261W + L262E S9E + N43R + N76D + A194P + N204D + V205I +Q206L + 1.1 1.1 1.1 Y209W + S212G + S216V + S259D + N261W + L262E S9E +N43R + N76D + A194P + Q206L + Y209W + L262E 1.1 1.1 1.1 S9E + N43R +N76D + A194P + Q206L + Y209W + S256D + 0.9 0.9 1.0 S259D + N261W + L262ES9E + N43R + N76D + A194P + Q206L + Y209W + T255E + 1.1 1.1 1.2 S256D +S259D + T260E + N261W + L262E S9E + N43R + N76D + V205I + Q206L +Y209W + S259D + 1.1 1.3 1.2 N261W + L262E S9E + N43R + N76D + G115W +H120V + A194P + Q206L + 0.9 1.0 1.1 Y209W + S259D + L262E S9E + N43R +N76D + G115W + H120V + P129D + A194P + 0.8 1.0 0.9 Q206L + Y209W +S259D + L262E S9E + N43R + N76D + G160P + A194P + N204D + V205I + 1.31.4 1.2 Q206L + Y209W + S212G + S216V + L262E S9E + N43R + N76D +G160P + S161E + A194P + N204D + 1.1 1.4 1.3 V205I + Q206L + Y209W +S212G + S216V + L262E S9E + N43R + N76D + G160P + S161E + A194P +N204D + 1.4 1.5 1.5 V205I + Q206L + Y209W + S212G + S216V + L262E +*275aH + *275bH S9E + N43R + N76D + N204D + V205I + Q206L + Y209W + 1.01.1 1.4 S259D + N261W + L262E S9E + N43R + N76D + G160P + S161E +V205I + Q206L + 1.1 1.2 1.3 Y209W + S259D + N261W + L262E S9E + N43R +N76D + H120V + Q182E + A194P + V205I + 1.0 1.1 1.1 Q206L + Y209W +S256D + N261W + L262E + *275aH + *275bH S9E + N43R + N76D + V205I +Q206L + Y209W + S216V + 0.8 1.0 1.2 S259D + N261W + L262E S9E + N43R +N76D + S161E + V205I + Q206L + Y209W + 0.9 1.0 1.5 S259D + N261W + L262ES9E + N43R + N76D + P131* + A194P + Q206L + Y209W + 1.5 1.4 1.4 S259D +L262E S9E + N43R + N76D + H120V + Q182E + A194P + V205I + 0.9 1.0 1.2Q206L + Y209W + S256D + N261W + L262E + *275aH + *275bH S9E + N43R +N76D + S161E + A194P + N204D + V205I + 0.9 1.2 1.2 Q206L + Y209W +S212G + S216V + L262E + *275aH + *275bH S9E + N43R + N76D + V205I +Q206L + Y209W + S212G + 1.0 1.2 1.5 S259D + N261W + L262E S9E + N43R +N76D + N204D + V205I + Q206L + Y209W + 1.1 1.2 1.3 S212G + S216V +S259D + N261W + L262EThe results Table 30 show that the Subtilisin309 variants showedimproved or on par wash performance compared to Subtilisin309 (SEQ IDNO: 2) on Blood/milk/ink at 25° C.

1. A liquid cleaning composition having a pH from about 7 to about 13comprising a protease variant having at least 60% identity with theamino acid sequence of SEQ ID NO: 1 or 2, and wherein said variantcomprises a substitution at one or more positions selected frompositions 218 and 259, wherein position numbers correspond to positionsin SEQ ID NO:
 1. 2. The liquid cleaning composition of claim 1, whereinthe composition has a pH from about 8 to about
 13. 3. The liquidcleaning composition of claim 1, wherein the composition has a pH fromabout 9 to about
 13. 4. The liquid cleaning composition of claim 1,wherein the composition has a pH from about 10 to about
 13. 5. Theliquid cleaning composition of claim 1, wherein the composition has a pHfrom about 11 to about
 13. 6. The liquid cleaning composition of claim1, wherein the protease variant comprises substitutions at position 218and position
 259. 7. The liquid cleaning composition of claim 2, whereinthe protease variant comprises substitutions at position 218 andposition
 259. 8. The liquid cleaning composition of claim 3, wherein theprotease variant comprises substitutions at position 218 and position259.
 9. The liquid cleaning composition of claim 4, wherein the proteasevariant comprises substitutions at position 218 and position
 259. 10.The liquid cleaning composition of claim 5, wherein the protease variantcomprises substitutions at position 218 and position
 259. 11. A liquidcleaning composition comprising: (a) from 0% to 20% wt of a detergentsurfactant (b) from 40% to 95% wt water; (c) from 1% to 30% wt of analkaline buffer system comprising an alkali metal silicate or an alkalimetal hydroxide or a mixture thereof, to provide a pH of from about 11to 13.5; and (d) from 0.001% to 10% wt of a protease variant, whereinsaid variant comprises a substitution at one or more positions selectedfrom positions 218 and 259, wherein position numbers correspond topositions in SEQ ID NO:
 1. 12. A liquid cleaning composition comprising:(a) from 0% to 20% wt of a detergent surfactant; (b) from 0.001% to 10%of a protease variant, wherein said variant comprises a substitution atone or more positions selected from positions 218 and 259, whereinposition numbers correspond to positions in SEQ ID NO: 1; and (c1) from20% to 95% wt water; and/or (c2) from 1% to 30% wt of an alkaline buffersystem comprising an alkali metal silicate or an alkali metal hydroxideor a mixture thereof, to provide a pH of from about 8 to 13.5.
 13. Aliquid cleaning composition having pH 10 or above, comprising: at least0.01 wt % protease, wherein the protease is a variant of a parentprotease and has an amino acid sequence which has at least 60% sequenceidentity to the amino acid sequence of SEQ ID NO: 2, and wherein saidprotease variant is characterized by having at least 10% higher residualactivity than the parent protease when measured after 4 hours at 40° C.in liquid detergent with pH 10, and wherein said variant comprises asubstitution at one or more positions selected from positions 218 and259, wherein position numbers correspond to positions in SEQ ID NO: 1.14. A liquid cleaning composition comprising: (a) from 20% to 95% wtwater; and (b) at least 0.01 wt % protease, wherein the protease is avariant of a parent protease and has an amino acid sequence which has atleast 60% sequence identity to the amino acid sequence of SEQ ID NO: 2,and wherein said protease variant is characterized by having at least10% higher residual activity than the parent protease when measuredafter 4 hours at 40° C. in liquid detergent with pH 10, and wherein saidvariant comprises a substitution at one or more positions selected frompositions 218 and 259, wherein position numbers correspond to positionsin SEQ ID NO:
 1. 15. A liquid cleaning composition comprising: (a) from0% to 20% wt of a detergent surfactant; (b) from 0.001% to 10% wt of aprotease variant, wherein said variant comprises a substitution at oneor more positions selected from positions 218 and 259, wherein positionnumbers correspond to positions in SEQ ID NO: 1; and (c1) from 20% to95% wt water; and/or (c2) from 1% to 30% wt of an alkaline buffer systemcomprising an alkali metal silicate or an alkali metal hydroxide or amixture thereof, to provide a pH of from about 11 to 13.5.